Methods for amplifying trichomonas vaginalis-derived nucleic acid

ABSTRACT

Oligonucleotides useful for determining the presence of  Trichomonas vaginalis  in a test sample. The oligonucleotides may be incorporated into detection probes, helper probes, capture probes and amplification oligonucleotides, and used in various combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/848,922, filed May 18, 2004, now pending, which claims the benefit ofU.S. Provisional Application No. 60/472,028, filed May 19, 2003, thecontents of each of which applications is hereby incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to detection probes, helper probes,capture probes, amplification oligonucleotides, nucleic acidcompositions, probe mixes, methods, and kits useful for determining thepresence of Trichomonas vaginalis in a test sample.

BACKGROUND OF THE INVENTION

Trichomonas vaginalis is protozoan parasite that causes trichomoniasis,one of the most common and treatable of the sexually transmitteddiseases. Trichomonas vaginalis is a relatively delicate pear-shapedtrophozoite that is typically 7 to 23 μm long by 5 to 12 μm wide. Theorganism has four anterior flagella and a fifth forming the outer edgeof a short undulating membrane. The anterior flagella propels theorganism through liquid in a jerky, rapid fashion, sometimes causing theorganism to rotate as it moves. Trichomonas vaginalis divides by binaryfission in the urogenital tract of those infected. The organism isclear, uncolored, or slightly grey in appearance under the microscope. Aslender rod, the axostyle, extends the length of the body and protrudesposteriorly. The nucleus is near-anterior and appears well-defined,containing many chromatin granules. The appearance of T. vaginalis isvery similar to that of other trichomonads, such as Trichomonas tenax,although only T. vaginalis is found in genitourinary tract infections.

Worldwide, T. vaginalis infects approximately 180 million people peryear, usually by direct person-to-person contact, making it the mostcommon sexually transmitted disease (STD) agent. In the United States,it is believed that T. vaginalis infects an estimated 5 million peopleannually. Despite its prevalence and geographic distribution, T.vaginalis has not been the focus of intensive study. Indeed, it is noteven listed as a “reportable disease” by the U.S. Centers for DiseaseControl, and there are no active control or prevention programs. Recentreports, however, suggest growing public health interest in thispathogen.

Infections in women are known to cause vaginitis, urethritis, andcervicitis. Severe infections are accompanied by a foamy,yellowish-green discharge with a foul odor, and small hemorrhagiclesions may also be present in the genitourinary tract. Complicationsinclude premature labor, low-birth weight offspring, premature ruptureof membranes, and post-abortion and post-hysterectomy infection. Anassociation with pelvic inflammatory disease, tubal infertility, andcervical cancer have been reported. Trichomonas vaginalis has also beenimplicated as a co-factor in the transmission of HIV and other STDagents. The organism can also be passed to neonates during passagethrough the birth canal.

In men, symptoms of trichomoniasis include urethral discharge, urethralstricture, epididymitis, the urge to urinate, and a burning sensationwith urination. In both men and women, infections with T. vaginalis areusually asymptomatic and self-limiting. It is estimated that, in women,10-50% of T. vaginalis infections are asymptomatic, with the proportionin men probably being even higher. That said, with many women theinfection becomes symptomatic and chronic, with periods of relief inresponse to therapy. Recurrence may be caused by re-infection from anasymptomatic sexual partner, or by failure of the standard course oftherapy (a regimen of the antibiotic metronidazole). And while T.vaginalis infections almost always occur in the genitourinary tract, onrare occasions they occur at ecotopic sites, and the parasite may berecovered from other areas of a patient's body.

As a result of suboptimal comparative laboratory methods and a focus onother STD sources, studies of T. vaginalis have often substantiallyunderestimated the prevalence of infection. Despite this, levels ofinfection typically have been high, with reported overall prevalencesranging from 3-58%, with an unweighted average across studies of 21%(Cu-Uvin et al. Clin. Infect. Dis. (2002) 34(10):1406-11). In studiesthat presented information on race/ethnicity, T. vaginalis infectionrates have been reported to be highest among African-Americans (Sorvilloet al. Emerg. Infect. Dis. (2001) 7(6):927-32). The following chartillustrates the trend reported by Sorvillo et al., with regard to theprevalence of infection in terms of the percentage of patients infectedwith trichomoniasis, chlamydia, and/or gonorrhea at certain healthclinics in Baltimore, Md. (B) and in New York, N.Y. (NY).

Patient Trichomoniasis Chlamydia Gonorrhea Year Number City (%) (%) (%)1996 213 NY 51 9 5 1994 372 NY 27 7 2 1994 1404 NY 20 15 No Data 1992279 B 26 21 14 1990-94 677 NY 22 6 1

Following exposure, the incubation period ranges from about 5 to 10days, although periods as short as 1 day to as many as 28 days have beenreported. If diagnosed, T. vaginalis infections can be readily treatedby orally administered antibiotics.

Given its relative prevalence and association with other STDs, there isincreasing interest in effectively diagnosing trichomoniasis.Conventional diagnostic methods for detecting T. vaginalis, however, arebased on direct examination, “wet mount” microscopy, or cell culture,each of which has its own shortcomings. With regard to direct patientexamination, other infections mimic the appearance and odor of thevaginal discharge. Accordingly, laboratory techniques such asmicroscopy, antibody detection, and cell culture are often used. Whileit is possible to detect T. vaginalis using a “wet mount” prepared bymixing vaginal secretions with saline on a slide and examining the slideunder a microscope for the presence of organisms having thecharacteristic size, shape, and motility of T. vaginalis, thesensitivity of such methods depends highly on the skill and experienceof the microscopist, as well as the time spent transporting specimen toa laboratory. Wet mount diagnosis has been found to be only 35-80% assensitive as other methods, such as cell culture, in detecting thepresence of T. vaginalis. Other direct methods, such as fluorescentantibody detection and enzyme-linked immunoassays, have also beendeveloped, as has a non-amplified, DNA probe-based method (Affirm,Becton Dickinson), although their sensitivities, as compared to cellculture, range from 70-90%. For these reasons, cell culture isconsidered the current “gold standard” for clinical detection of T.vaginalis. Due to its relatively delicate nature, however, the organismis technically challenging, and typically requires up to 7 days formaximum sensitivity. Even then, the sensitivity of cell culture methodsis estimated to be only about 85-95% due to problems associated withtime lapses between sample recovery and culture inoculation, maintainingproper incubation conditions, visualizing low numbers of the organismand/or the motility of the protozoa.

Given the human health implications of trichomoniasis and relativeinability of existing clinical laboratory methods to selectively andsensitively detect T. vaginalis from a test sample, a need clearlyexists for a sensitive and specific assay which can be used to determinethe presence of T. vaginalis in a particular sample of biologicalmaterial.

SUMMARY OF THE INVENTION

The present invention provides a solution to the clinical need for asensitive assay specific for T. vaginalis by featuring oligonucleotidesthat are useful for determining whether T. vaginalis is present in atest sample, such as a genitourinary specimen. The featuredoligonucleotides may be contained in detection probes, helper probes,capture probes and/or amplification oligonucleotides that are useful fordetecting, immobilizing and/or amplifying T. vaginalis target nucleicacid present in a test sample.

In one embodiment, detection probes are provided that can preferentiallyhybridize to a target region present in nucleic acid derived from T.vaginalis to form a detectable probe:target hybrid indicating thepresence of T. vaginalis. In preferred embodiments, the inventionprovides a detection probe for determining whether T. vaginalis ispresent in a test sample derived from a biological material, preferablytaken from the genitourinary tract of a patient. The detection probecontains a target binding region having an at least 10 contiguous basesequence that is at least about 80%, 90% or 100% complementary to an atleast 10 contiguous base region present in a target sequence selectedfrom the group consisting of:

SEQ ID NO:1: gccgaagtccttcggttaaagttctaattggg, SEQ ID NO:2:gccgaaguccuucgguuaaaguucuaauuggg, SEQ ID NO:3:cccaattagaactttaaccgaaggacttcggc, and SEQ ID NO:4:cccaauuagaacuuuaaccgaaggacuucggc.

In another preferred embodiment, the present invention provides adetection probe which contains a target binding region having an atleast 10 contiguous base sequence that is at least about 80%, 90% or100% complementary to an at least 10 contiguous base region present in atarget sequence selected from the group consisting of:

SEQ ID NO:5: ccattggtgccttttggtactgtggatagg, SEQ ID NO:6:ccauuggugccuuuugguacuguggauagg, SEQ ID NO:7:cctatccacagtaccaaaaggcaccaatgg, SEQ ID NO:8:ccuauccacaguaccaaaaggcaccaaugg, SEQ ID NO:9: ttccattggtgccttttggtactgtg,SEQ ID NO:10: uuccauuggugccuuuugguacugug, SEQ ID NO:11:cacagtaccaaaaggcaccaatggaa, SEQ ID NO:12: cacaguaccaaaaggcaccaauggaa,SEQ ID NO:13: ccattggtgccttttggtactgtggat, SEQ ID NO:14:ccauuggugccuuuugguacuguggau, SEQ ID NO:15: atccacagtaccaaaaggcaccaatgg,and SEQ ID NO:16: auccacaguaccaaaaggcaccaaugg.The core region targeted by this preferred detection probe is selectedfrom the group consisting of:

SEQ ID NO:17: ccattggtgccttttggtactgtg, SEQ ID NO:18:ccauuggugccuuuugguacugug, SEQ ID NO:19: cacagtaccaaaaggcaccaatgg, andSEQ ID NO:20: cacaguaccaaaaggcaccaaugg.

Detection probes according to the invention preferentially hybridize tothe target nucleic acid and not to nucleic acid derived from non-T.vaginalis organisms present in a test sample under stringenthybridization conditions. In particular, the detection probes of thepresent invention preferentially hybridize to the target nucleic acidand not to nucleic acid derived from Trichomonas tenax, which isconsidered to be the most closely related organism to T. vaginalis.Trichomonas tenax can be obtained from the American Type CultureCollection in Manassas, Va. as ATCC No. 30207.

In the present invention, the detection probe may have a target bindingregion of any length suitable to achieve the desired selectivity andspecificity for T. vaginalis-derived nucleic acid. The base sequence ofa detection probe according to the present invention is preferably up to100 bases in length, more preferably from 10 to 50 bases in length, andmost preferably from 18 to 35 bases in length. In a preferredembodiment, the detection probe contains a target binding region havingan at least 15 contiguous base sequence which is at least about 80%, 90%or 100% complementary to an at least 15 contiguous base region presentin the target sequence. Preferably, the target binding region of thedetection probe comprises a base sequence which is fully complementaryto the target sequence. More preferably, the base sequence of the targetbinding region of the detection probe is at least about 80%, 90% or 100%complementary to the target sequence.

Most preferably, the base sequence of the detection probe is at leastabout 80%, 90% or 100% complementary to the target sequence.

The target binding region may consist of deoxyribonucleic acid (DNA),ribonucleic acid (RNA), a combination DNA and RNA, or it may be anucleic acid analog (e.g., a peptide nucleic acid) or contain one ormore modified nucleosides (e.g., a ribonucleoside having a 2′-O-methylsubstitution to the ribofuranosyl moiety). The target binding region mayadditionally include molecules that do not hydrogen bond to adenine,cytosine, guanine, thymine or uracil, provided such molecules do notinterfere with the ability of the detection probe to selectively andspecifically bind to nucleic acid derived from T. vaginalis in the testsample. Such molecules could include, by way of example, abasicnucleotides or universal base analogues, such as 5-nitroindole, providedsuch molecules do not significantly affect duplex stability. See, e.g.,Guo et al., “Artificial Mismatch Hybridization,” U.S. Pat. No.5,780,233, the contents of which are hereby incorporated by referenceherein.

A detection probe of the present invention may include one or more basesequences in addition to the base sequence of the target binding regionwhich do not stably bind to nucleic acid derived from T. vaginalis understringent hybridization conditions. An additional base sequence may becomprised of any desired base sequence, so long as it does not stablybind to nucleic acid derived from the T. vaginalis under stringenthybridization conditions or prevent stable hybridization of the probe tothe target nucleic acid. By way of example, an additional base sequencemay constitute the immobilized probe binding region of a capture probe,where the immobilized probe binding region is comprised of, for example,a 3′ poly dA (adenine) region which hybridizes under stringenthybridization conditions to a 5′ poly dT (thymine) region of apolynucleotide bound directly or indirectly to a solid support. Anadditional base sequence might also be a 5′ sequence recognized by a RNApolymerase or which enhances initiation or elongation by a RNApolymerase (e.g., a T7 promoter). More than one additional base sequencemay be included if the first sequence is incorporated into, for example,a self-hybridizing probe (i.e., a probe having distinct base regionscapable of hybridizing to each other in the absence of a target sequenceunder the conditions of an assay), such as a “molecular beacon” probe.Molecular beacons are disclosed by Tyagi et al., “Detectably LabeledDual Conformation Oligonucleotide Probes, Assays and Kits,” U.S. Pat.No. 5,925,517 (the contents of which are hereby incorporated byreference herein), and include a target binding region which is boundedby or overlaps with two base sequences having regions, referred to as“stems” or “arms,” which are at least partially complementary to eachother. A more detailed description of molecular beacons is providedinfra in the section entitled “Detection Probes to Trichomonas vaginalisRibosomal Nucleic Acid.” An additional base sequence may be joineddirectly to the target binding region or, for example, by means of anon-nucleotide linker (e.g., polyethylene glycol or an abasic region).

While not required, detection probes of the present invention preferablyinclude at least one detectable label or group of interacting labels.The label may be any suitable labeling substance, including but notlimited to a radioisotope, an enzyme, an enzyme cofactor, an enzymesubstrate, a dye, a hapten, a chemiluminescent molecule, a fluorescentmolecule, a phosphorescent molecule, an electrochemiluminescentmolecule, a chromophore, a base sequence region that is unable to stablyhybridize to the target nucleic acid under the stated conditions, andmixtures of these. In one particularly preferred embodiment, the labelis an acridinium ester (AE), preferably 4-(2-succinimidyloxycarbonylethyl)-phenyl-10-methylacridinium-9-carboxylate fluorosulfonate(hereinafter referred to as “standard AE”). Groups of interacting labelsuseful with a probe pair (see, e.g., Morrison, “Competitive HomogeneousAssay,” U.S. Pat. No. 5,928,862) or a self-hybridizing probe (see, e.g.,Tyagi et al., U.S. Pat. No. 5,925,517) include, but are not limited to,enzyme/substrate, enzyme/cofactor, luminescent/quencher,luminescent/adduct, dye dimers and Forrester energy transfer pairs. Aninteracting luminescent/quencher pair, such as fluoroscein and DABCYL,is particularly preferred.

In a further embodiment, the present invention contemplates probe mixesthat are useful for determining whether T. vaginalis is present in atest sample. The probe mix may comprise, for example, one of theabove-described T. vaginalis detection probes and a helper probe. Thebase sequence of a helper probe according to the present invention ispreferably up to 100 bases in length, more preferably from 10 to 50bases in length, and most preferably from 18 to 35 bases in length. Thehelper probe preferably contains an at least 10 contiguous base regionwhich is at least about 80%, 90% or 100% complementary to an at least 10contiguous base region present in a target sequence selected from thegroup consisting of:

SEQ ID NO:21: gctaacgagcgagattatcgccaattatttacttt, SEQ ID NO:22:gcuaacgagcgagauuaucgccaauuauuuacuuu, SEQ ID NO:23:aaagtaaataattggcgataatctcgctcgttagc, SEQ ID NO:24:aaaguaaauaauuggcgauaaucucgcucguuagc, SEQ ID NO:25:actccctgcgattttagcaggtggaagagg, SEQ ID NO:26:acucccugcgauuuuagcagguggaagagg, SEQ ID NO:27:cctcttccacctgctaaaatcgcagggagt, and SEQ ID NO:28:ccucuuccaccugcuaaaaucgcagggagu.Helper probes according to the present invention need not exhibitspecificity for the target sequence in a test sample. In a preferredembodiment, the helper probe comprises an at least 15 contiguous basesequence which is at least about 80%, 90% or 100% complementary to an atleast 15 contiguous base region present in the target sequence.Preferably, the helper probe comprises a base sequence which is fullycomplementary to the target sequence. The base sequence of the helperprobe of the present invention is most preferably at least about 80%,90% or 100% complementary to the target sequence. In a preferred probemix, the detection probe comprises an at least 10 contiguous base regionwhich is at least about 80% complementary to an at least 10 contiguousbase region present in a sequence selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

The invention also contemplates compositions comprising stable nucleicacid duplexes formed between any of the above-described detection probesand/or helper probes and the target nucleic acids for the probes understringent hybridization conditions.

In another embodiment of the present invention, a capture probe isprovided for specifically isolating and purifying target nucleic acidderived from T. vaginalis present in a test sample. The capture probeincludes a target binding region that stably binds to nucleic acidderived from T. vaginalis under assay conditions and which has an atleast 10 contiguous base region which is at least about 80%, 90% or 100%complementary to an at least 10 contiguous base region present in atarget sequence selected from the group consisting of:

SEQ ID NO:29: atatccacgggtagcagcaggc, SEQ ID NO:30:auauccacggguagcagcaggc, SEQ ID NO:31: gcctgctgctacccgtggatat, and SEQ IDNO:32: gccugcugcuacccguggauau.

The base sequence of the target binding region of a capture probeaccording to the present invention is preferably up to 100 bases inlength, more preferably from 10 to 50 bases in length, and mostpreferably from 18 to 35 bases in length. In a preferred embodiment, thetarget binding region of the capture probe comprises an at least 15contiguous base sequence which is at least about 80%, 90% or 100%complementary to an at least 15 contiguous base region present in thetarget sequence. Preferably, the target binding region of the captureprobe comprises a base sequence fully complementary to the targetsequence. The base sequence of the target binding region of the captureprobe of the present invention is more preferably at least about 80%,90% or 100% complementary to the target sequence. In a most preferredembodiment, the base sequence of the target binding region of thecapture probe is at least about 80%, 90% or 100% complementary to thetarget sequence, and the capture probe does not include any other basesequences which stably hybridize to nucleic acid derived from T.vaginalis under assay conditions.

Capture probes according to the present invention may be immobilized ona solid support by means of ligand-ligate binding pairs, such asavidin-biotin linkages, but preferably include an immobilized probebinding region. The immobilized probe binding region of the preferredcapture probes is comprised of any base sequence capable of stablyhybridizing under assay conditions to an oligonucleotide that is boundto a solid support present in a test sample. Preferably, the immobilizedprobe binding region is a poly dA, homopolymer tail positioned at the 3′end of the capture probe. In this embodiment, oligonucleotides bound tothe solid support would include 5′ poly dT tails of sufficient length tostably bind to the poly dA tails of the capture probes under assayconditions. In a preferred embodiment, the immobilized probe bindingregion includes a poly dA tail which is about 30 adenines in length, andthe capture probe includes a spacer region which is about 3 thymines inlength for joining together the target binding region and theimmobilized probe binding region.

The present invention also features amplification oligonucleotidesuseful for determining the presence of T. vaginalis in an amplificationassay. In a preferred embodiment, the invention provides at least oneamplification oligonucleotide for amplifying nucleic acid derived fromT. vaginalis present in a test sample, where the amplificationoligonucleotide has a target binding region that preferably contains anat least 10 contiguous base region which is at least about 80%, 90% or100% complementary to an at least 10 contiguous base region present in atarget sequence selected from the group consisting of:

SEQ ID NO:33: gcgttgattcagctaacgagcgagattatcgcc, SEQ ID NO:34:gcguugauucagcuaacgagcgagauuaucgcc, SEQ ID NO:35:ggcgataatctcgctcgttagctgaatcaacgc, SEQ ID NO:36:ggcgauaaucucgcucguuagcugaaucaacgc, SEQ ID NO:37:ctgcgattttagcaggtggaagagggtagcaataaca ggtccgtgatgcc, SEQ ID NO:38:cugcgauuuuagcagguggaagaggguagcaauaaca gguccgugaugcc, SEQ ID NO:39:ggcatcacggacctgttattgctaccctcttccacct gctaaaatcgcag, and SEQ ID NO:40:ggcaucacggaccuguuauugcuacccucuuccaccu gcuaaaaucgcag.More preferably, the target sequence of the amplificationoligonucleotide is selected from the group consisting of:

SEQ ID NO:41: gcgttgattcagctaacgagcg, SEQ ID NO:42:gcguugauucagcuaacgagcg, SEQ ID NO:43: cgctcgttagctgaatcaacgc, SEQ IDNO:44: cgcucguuagcugaaucaacgc, SEQ ID NO:45: gctaacgagcgagattatcgcc, SEQID NO:46: gcuaacgagcgagauuaucgcc, SEQ ID NO:47: ggcgataatctcgctcgttagc,SEQ ID NO:48: ggcgauaaucucgcucguuagc, SEQ ID NO:49:ctgcgattttagcaggtggaagagg, SEQ ID NO:50: cugcgauuuuagcagguggaagagg, SEQID NO:51: cctcttccacctgctaaaatcgcag, SEQ ID NO:52:ccucuuccaccugcuaaaaucgcag, SEQ ID NO:53: gcaataacaggtccgtgatgcc, SEQ IDNO:54: gcaauaacagguccgugaugcc, SEQ ID NO:55: ggcatcacggacctgttattgc, andSEQ ID NO:56: ggcaucacggaccuguuauugc.

In another preferred embodiment, the at least one amplificationoligonucleotide for amplifying nucleic acid derived from T. vaginalispresent in a test sample has a target binding region that preferablycontains an at least 10 contiguous base region which is at least about80%, 90% or 100% complementary to an at least 10 contiguous base regionpresent in a target sequence selected from the group consisting of:

SEQ ID NO:57: ggtagcagcaggcgcgaaactttcccactcgagactt tcggaggaggtaat, SEQID NO:58: gguagcagcaggcgcgaaacuuucccacucgagacuu ucggaggagguaau, SEQ IDNO:59: attacctcctccgaaagtctcgagtgggaaagtttcg cgcctgctgctacc, SEQ IDNO:60: auuaccuccuccgaaagucucgagugggaaaguuucg cgccugcugcuacc, SEQ IDNO:61: accgtaccgaaacctagcagagggccagtctggtgcc agcagc, SEQ ID NO:62:accguaccgaaaccuagcagagggccagucuggugcc agcagc, SEQ ID NO:63:gctgctggcaccagactggccctctgctaggtttcgg tacggt, and SEQ ID NO:64:gcugcuggcaccagacuggcccucugcuagguuucgg uacggu.More preferably, the target sequence of the amplificationoligonucleotide is selected from the group consisting of:

SEQ ID NO:65: ggtagcagcaggcgcg, SEQ ID NO:66: gguagcagcaggcgcg, SEQ IDNO:67: cgcgcctgctgctacc, SEQ ID NO:68: cgcgccugcugcuacc, SEQ ID NO:69:ccactcgagactttcggagg, SEQ ID NO:70: ccacucgagacuuucggagg, SEQ ID NO:71:cctccgaaagtctcgagtgg, SEQ ID NO:72: ccuccgaaagucucgagugg, SEQ ID NO:73:gagactttcggaggaggtaat, SEQ ID NO:74: gagacuuucggaggagguaau, SEQ IDNO:75: attacctcctccgaaagtctc, SEQ ID NO:76: auuaccuccuccgaaagucuc, SEQID NO:77: accgtaccgaaacctagcagagg, SEQ ID NO:78:accguaccgaaaccuagcagagg, SEQ ID NO:79: cctctgctaggtttcggtacggt, SEQ IDNO:80: ccucugcuagguuucgguacggu, SEQ ID NO:81: cgaaacctagcagagggccagtc,SEQ ID NO:82: cgaaaccuagcagagggccaguc, SEQ ID NO:83:gactggccctctgctaggtttcg, SEQ ID NO:84: gacuggcccucugcuagguuucg, SEQ IDNO:85: ccagtctggtgccagcagc, SEQ ID NO:86: ccagucuggugccagcagc, SEQ IDNO:87: gctgctggcaccagactgg, and SEQ ID NO:88: gcugcuggcaccagacugg.

Amplification oligonucleotides of the present invention have a targetbinding region that is preferably from 18 to 40 bases in length. In apreferred embodiment, the amplification oligonucleotide contains atarget binding region having an at least 15 contiguous base sequencewhich is at least about 80%, 90% or 100% complementary to an at least 15contiguous base region present in the target sequence. Preferably, thetarget binding region of the amplification oligonucleotide comprises abase sequence which is fully complementary to the target sequence. Morepreferably, the base sequence of the target binding region of theamplification oligonucleotide is at least about 80%, 90% or 100%complementary to the target sequence, and the amplificationoligonucleotide does not include any other base sequences which stablyhybridize to nucleic acid derived from T. vaginalis under amplificationconditions. The amplification oligonucleotide optionally includes a 5′sequence which is recognized by a RNA polymerase or which enhancesinitiation or elongation by RNA polymerase. The T7 promoter sequence ofSEQ ID NO:89: aatttaatacgactcactatagggaga is preferred, although otherpromoter sequences may be employed.

The invention further contemplates an amplification oligonucleotidewhich, when contacted with a nucleic acid polymerase under amplificationconditions, will bind to or cause extension through a nucleic acidregion having a base sequence selected from the group consisting of: SEQID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ IDNO:56. In an alternative embodiment, the amplification oligonucleotidebinds to or extends through a nucleic acid region having a base sequenceselected from the group consisting of: SEQ ID NO:65, SEQ ID NO:66, SEQID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87 and SEQ ID NO:88. The base sequence of an amplificationoligonucleotide of such embodiments consists of a target binding regionup to 40 bases in length and an optional 5′ sequence which is recognizedby a RNA polymerase or which enhances initiation or elongation by RNApolymerase (e.g., T7 promoter of SEQ ID NO:89).

Amplification oligonucleotides of the present invention are preferablyemployed in sets of at least two amplification oligonucleotides. Onepreferred set includes a first amplification oligonucleotide having atarget binding region which contains an at least 10 contiguous baseregion which is at least about 80% complementary to an at least 10contiguous base region present in a target sequence selected from thegroup consisting of: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ IDNO:36. More preferably, the target sequence of the first amplificationoligonucleotide is selected from the group consisting of: SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47 and SEQ ID NO:48. The second amplification oligonucleotideof this preferred set has a target binding region that contains an atleast 10 contiguous base region which is at least about 80%complementary to an at least 10 contiguous base region present in atarget sequence selected from the group consisting of: SEQ ID NO:37, SEQID NO:38, SEQ ID NO:39 and SEQ ID NO:40. More preferably, the targetsequence of the second amplification oligonucleotide is selected fromthe group consisting of: SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56.Other structural embodiments of the first and second amplificationoligonucleotides are those set forth above for individual amplificationoligonucleotides. It is preferred that at least one member of the set ofamplification oligonucleotides include a 5′ sequence which is recognizedby a RNA polymerase or which enhances initiation or elongation by RNApolymerase (e.g., T7 promoter of SEQ ID NO:89).

Another set of preferred amplification oligonucleotides includes a firstamplification oligonucleotide having a target binding region thatcontains an at least 10 contiguous base region which is at least about80% complementary to an at least 10 contiguous base region present in atarget sequence selected from the group consisting of: SEQ ID NO:57, SEQID NO:58, SEQ ID NO:59 and SEQ ID NO:60. More preferably, the targetsequence of the first amplification oligonucleotide is selected from thegroup consisting of: SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76. The secondamplification oligonucleotide of this preferred set has a target bindingregion that contains an at least 10 contiguous base region which is atleast about 80% complementary to an at least 10 contiguous base regionpresent in a target sequence selected from the group consisting of: SEQID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64. More preferably,the target sequence of the second amplification oligonucleotide isselected from the group consisting of: SEQ ID NO:77, SEQ ID NO:78, SEQID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88. Otherstructural embodiments of the first and second amplificationoligonucleotides are those set forth above for individual amplificationoligonucleotides. It is preferred that at least one member of the set ofamplification oligonucleotides include a 5′ sequence which is recognizedby a RNA polymerase or which enhances initiation or elongation by RNApolymerase (e.g., T7 promoter of SEQ ID NO:89).

The invention additionally contemplates compositions comprising stablenucleic acid duplexes formed between any of the above-describedamplification oligonucleotides and the target nucleic acids for theamplification oligonucleotides under amplification conditions.

In yet another embodiment of the present invention, a set ofoligonucleotides is provided for determining the presence of T.vaginalis in a test sample, where each member of the set has a targetbinding region that contains an at least 10 contiguous base region whichis at least about 80% complementary to an at least 10 contiguous baseregion present in a target sequence selected from the group consistingof:

SEQ ID NO:90: gcgttgattcagctaacgagcgagattatcgccaattatttactttgccgaagtccttcggttaaagttctaattgggactccctgcgattttagcaggtggaagagggta gcaataacaggtccgtgatgcc, SEQ IDNO:91: gcguugauucagcuaacgagcgagauuaucgccaauuauuuacuuugccgaaguccuucgguuaaaguucuaauugggacucccugcgauuuuagcagguggaagagggua gcaauaacagguccgugaugcc, SEQ IDNO:92: ggcatcacggacctgttattgctaccctcttccacctgctaaaatcgcagggagtcccaattagaactttaaccgaaggacttcggcaaagtaaataattggcgataatct cgctcgttagctgaatcaacgc, and SEQ IDNO:93: ggcaucacggaccuguuauugcuacccucuuccaccugcuaaaaucgcagggagucccaauuagaacuuuaaccgaaggacuucggcaaaguaaauaauuggcgauaaucu cgcucguuagcugaaucaacgc.In a preferred embodiment, the set of amplification oligonucleotidesincludes at least one detection probe, preferably one of theabove-described detection probes, which preferentially hybridizes to thetarget sequence and not to nucleic acid derived from non-T. vaginalisorganisms present in a test sample under stringent hybridizationconditions. In another preferred embodiment, the set of oligonucleotidesincludes at least two oligonucleotides, preferably including one of theabove-described detection probes and a helper probe which hybridizes tothe target sequence under stringent hybridization conditions, therebyfacilitating hybridization of the detection probe to the targetsequence, where the helper probe is preferably one of theabove-described helper probes. In yet another preferred embodiment, theset of oligonucleotides includes at least three oligonucleotides,preferably including one of the above-described detection probes and apair of amplification oligonucleotides capable of amplifying all or aportion of the target sequence under amplification conditions,preferably including at least one of the above-described amplificationoligonucleotides. And, in a particularly preferred embodiment, eachmember of the set of oligonucleotides contains an a target bindingregion which is fully complementary to a sequence contained within thetarget sequence, and none of the oligonucleotides includes any otherbase sequences which stably hybridize to nucleic acid derived from T.vaginalis under assay conditions.

In still another embodiment of the present invention, a set ofoligonucleotides is provided for determining the presence of T.vaginalis in a test sample, where each member of the set has a targetbinding region that contains an at least 10 contiguous base region whichis at least about 80% complementary to an at least 10 contiguous baseregion present in a target sequence selected from the group consistingof:

SEQ ID NO:94: ggtagcagcaggcgcgaaactttcccactcgagactttcggaggaggtaatgaccagttccattggtgccttttggtactgtggataggggtacggttttccaccgtaccgaaacctagcagagggccagtctggtgccagcagc, SEQ ID NO:95:gguagcagcaggcgcgaaacuuucccacucgagacuuucggaggagguaaugaccaguuccauuggugccuuuugguacuguggauagggguacgguuuuccaccguaccgaaaccuagcagagggccagucuggugccagcagc, SEQ ID NO:96:gctgctggcaccagactggccctctgctaggtttcggtacggtggaaaaccgtacccctatccacagtaccaaaaggcaccaatggaactggtcattacctcctccgaaagtctcgagtgggaaagtttcgcgcctgctgctacc, and SEQ ID NO:97:gcugcuggcaccagacuggcccucugcuagguuucgguacgguggaaaaccguaccccuauccacaguaccaaaaggcaccaauggaacuggucauuaccuccuccgaaagucucgagugggaaaguuucgcgccugcugcuacc.In one preferred embodiment, the set of amplification oligonucleotidesincludes at least one detection probe, preferably one of theabove-described detection probes, which preferentially hybridizes to thetarget sequence and not to nucleic acid derived from non-T. vaginalisorganisms present in a test sample under stringent hybridizationconditions. In another preferred embodiment, the set of oligonucleotidesincludes at least three oligonucleotides, preferably including one ofthe above-described detection probes and a pair of amplificationoligonucleotides capable of amplifying all or a portion of the targetsequence under amplification conditions, preferably including at leastone of the above-described amplification oligonucleotides. And, in aparticularly preferred embodiment, each member of the set ofoligonucleotides contains an a target binding region which is fullycomplementary to a sequence contained within the target sequence, andnone of the oligonucleotides includes any other base sequences whichstably hybridize to nucleic acid derived from T. vaginalis under assayconditions.

The present invention further features methods for determining whetherT. vaginalis is present in a test sample. In certain embodiments, theinvention provides methods for determining whether T. vaginalis ispresent in a test sample, where such methods comprise the steps of: (a)contacting the test sample with one of the above-described detectionprobes for detecting T. vaginalis under conditions permitting the probeto preferentially hybridize to a target nucleic acid derived from T.vaginalis, thereby forming a probe:target hybrid stable for detection;and (b) determining whether the hybrid is present in the test sample asan indication of the presence or absence of T. vaginalis in the testsample. This method may further include the step of quantifying theamount of hybrid present in the test sample as a means for estimatingthe amount of T. vaginalis present in the test sample.

The methods for determining whether T. vaginalis is present in a testsample, or the amount of T. vaginalis present in a test sample, mayfurther include the step of contacting the test sample with one of theabove-described helper probes for facilitating hybridization of thedetection probe to a target sequence and/or one of the above-describedcapture probes for isolating and purifying a target nucleic acid and/orone of the above-described amplification oligonucleotides appropriatefor amplifying a target region present in nucleic acid derived from T.vaginalis, as desired.

The invention also contemplates methods for amplifying a target sequencecontained in nucleic acid derived from T. vaginalis present in a testsample, where the method comprises the steps of: (a) contacting the testsample with at least one of the above-described amplificationoligonucleotides; and (b) exposing the test sample to conditionssufficient to amplify the target sequence. Preferred amplificationmethods will include a set of at least two of the above-describedamplification oligonucleotides.

In preferred embodiments, the methods for amplifying a target nucleicacid sequence present in nucleic acid derived from T. vaginalis willfurther include the steps of: (a) contacting the test sample with adetection probe which preferentially hybridizes to the target sequenceor its complement under stringent hybridization conditions, therebyforming a probe:target hybrid stable for detection; and (b) determiningwhether the hybrid is present in the test sample as an indication of thepresence or absence of T. vaginalis in the test sample. Theabove-described detection probes are preferred for these methods.

The invention also contemplates kits for determining whether T.vaginalis is present in a test sample. These kits include at least oneof the above-described detection probes specific for a target sequencederived from T. vaginalis and optionally include written instructionsfor determining the presence or amount of T. vaginalis in a test sample.In another embodiment, the kits further include the above-describedhelper probe for aiding hybridization of the detection probe to thetarget sequence. In a further embodiment, the kits also include at leastone of the above-described amplification oligonucleotides appropriatefor amplifying the target sequence or its complement. In yet anotherembodiment, the kits further include the above-described capture probefor separating the target sequence from other components of the testsample prior to amplifying or directly detecting the target sequence orits complement. In still another embodiment, the kits additionallyinclude at least two members of a group made up of one or more of theabove-described amplification oligonucleotides, the above-describedcapture probe and the above-described helper probe.

The invention also contemplates kits for amplifying a target sequencepresent in nucleic acid derived from T. vaginalis which include at leastone of the above-described amplification oligonucleotides and optionallyinclude written instructions for amplifying nucleic acid derived from T.vaginalis. In another embodiment, the kits further include theabove-described capture probe for separating the target sequence fromother components of the test sample prior to amplifying the targetsequence.

Those skilled in the art will appreciate that the detection probes ofthe present invention may be used as amplification oligonucleotides orcapture probes, the amplification oligonucleotides of the presentinvention may be used as helper probes or capture probes, and the helperprobes of the present invention may be used as amplificationoligonucleotides or capture probes, depending upon the degree ofspecificity required.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes oligonucleotides targeted to nucleicacids derived from T. vaginalis which are particularly useful fordetermining the presence or absence of T. vaginalis in a test sample.The oligonucleotides can aid in detecting T. vaginalis in differentways, such as by functioning as detection probes, helper probes, captureprobes and/or amplification oligonucleotides. Detection probes of thepresent invention can preferentially hybridize to a target nucleic acidsequence present in a target nucleic acid derived from T. vaginalisunder stringent hybridization conditions to form detectable duplexeswhich indicate the presence of T. vaginalis in a test sample. Probes ofthe present invention are believed to be capable of distinguishingbetween T. vaginalis and its known closest phylogenetic neighbor. Helperprobes of the present invention can hybridize to a target nucleic acidsequence present in nucleic acid derived from T. vaginalis understringent hybridization conditions and can be used to enhance theformation of detection probe:target nucleic acid duplexes. Captureprobes of the present invention can hybridize to a target nucleic acidsequence present in nucleic acid derived from T. vaginalis under assayconditions and can be used to separate target nucleic acid from othercomponents of a clinical specimen. Amplification oligonucleotides of thepresent invention can hybridize to a target nucleic acid sequencepresent in nucleic acid derived from T. vaginalis under amplificationconditions and can be used, for example, as primers in amplificationreactions to generate multiple copies of T. vaginalis-derived nucleicacid. The probes and amplification oligonucleotides can be used inassays for the detection and/or quantitation of T. vaginalis in a testsample.

A. Definitions

The following terms have the indicated meanings in the specificationunless expressly indicated to have a different meaning.

By “sample” or “test sample” is meant any substance suspected ofcontaining a target organism or nucleic acid derived from the targetorganism. The substance may be, for example, an unprocessed clinicalspecimen, such as a genitourinary tract specimen, a buffered mediumcontaining the specimen, a medium containing the specimen and lyticagents for releasing nucleic acid belonging to the target organism, or amedium containing nucleic acid derived from the target organism whichhas been isolated and/or purified in a reaction receptacle or on areaction material or device. In the claims, the terms “sample” and “testsample” may refer to specimen in its raw form or to any stage ofprocessing to release, isolate and purify nucleic acid derived fromtarget organisms in the specimen. Thus, within a method of use claim,each reference to a “sample” or “test sample” may refer to a substancesuspected of containing nucleic acid derived from the target organism ororganisms at different stages of processing and is not limited to theinitial form of the substance in the claim.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence.

By “target nucleic acid sequence,” “target sequence” or “target region”is meant a specific deoxyribonucleotide or ribonucleotide sequencecomprising all or part of the nucleotide sequence of a single-strandednucleic acid molecule.

By “oligonucleotide” or “oligomer” is meant a polymer made up of two ormore nucleoside subunits or nucleobase subunits coupled together. Theoligonucleotide may be DNA and/or RNA and analogs thereof. The sugargroups of the nucleoside subunits may be ribose, deoxyribose and analogsthereof, including, for example, ribonucleosides having a2′-O-methylsubstitution to the ribofuranosyl moiety. (Oligonucleotidesincluding nucleoside subunits having 2′ substitutions and which areuseful as detection probes, helper probes, capture probes and/oramplification oligonucleotides are disclosed by Becker et al., “Methodfor Amplifying Target Nucleic Acids Using Modified Primers,” U.S. Pat.No. 6,130,038.) The nucleoside subunits may be joined by linkages suchas phosphodiester linkages, modified linkages, or by non-nucleotidemoieties which do not prevent hybridization of the oligonucleotide toits complementary target nucleic acid sequence. Modified linkagesinclude those linkages in which a standard phosphodiester linkage isreplaced with a different linkage, such as a phosphorothioate linkage ora methylphosphonate linkage. The nucleobase subunits may be joined, forexample, by replacing the natural deoxyribose phosphate backbone of DNAwith a pseudo-peptide backbone, such as a 2-aminoethylglycine backbonewhich couples the nucleobase subunits by means of a carboxymethyl linkerto the central secondary amine. (DNA analogs having a pseudo-peptidebackbone are commonly referred to as “peptide nucleic acids” or “PNA,”and are disclosed by Nielsen et al., “Peptide Nucleic Acids,” U.S. Pat.No. 5,539,082.) Other non-limiting examples of oligonucleotides oroligomers contemplated by the present invention include nucleic acidanalogs containing bicyclic and tricyclic nucleoside and nucleotideanalogs referred to as “Locked Nucleic Acids,” “Locked NucleosideAnalogues” or “LNA.” (Locked Nucleic Acids are disclosed by Wang,“Conformationally Locked Nucleosides and Oligonucleotides,” U.S. Pat.No. 6,083,482; Imanishi et al., “Bicyclonucleoside and OligonucleotideAnalogues,” U.S. Pat. No. 6,268,490; and Wengel et al., “OligonucleotideAnalogues,” U.S. Pat. No. 6,670,461.) Any nucleic acid analog iscontemplated by the present invention, provided that the modifiedoligonucleotide can hybridize to a target nucleic acid under stringenthybridization conditions or amplification conditions. In the case ofdetection probes, the modified oligonucleotides must also be capable ofpreferentially hybridizing to the target nucleic acid under stringenthybridization conditions.

Oligonucleotides of a defined sequence may be produced by techniquesknown to those of ordinary skill in the art, such as by chemical orbiochemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules, e.g., bacterial or retroviralvectors. As intended by this disclosure, an oligonucleotide does notconsist of wild-type chromosomal DNA or the in vivo transcriptionproducts thereof. One use of an oligonucleotide is as a detection probe.Oligonucleotides may also be used as helper probes, capture probes andamplification oligonucleotides.

By “detection probe” or “probe” is meant a structure comprising anoligonucleotide having a base sequence sufficiently complementary to itstarget nucleic acid sequence to form a probe:target hybrid stable fordetection under stringent hybridization conditions. As would beunderstood by someone having ordinary skill in the art, theoligonucleotide is an isolated nucleic acid molecule, or an analogthereof, in a form not found in nature without human intervention (e.g.,recombined with foreign nucleic acid, isolated, or purified to someextent). The probes of this invention may have additional nucleosides ornucleobases complementary to nucleotides outside of the targeted regionso long as such nucleosides or nucleobases do not prevent hybridizationunder stringent hybridization conditions and, in the case of detectionprobes, do not prevent preferential hybridization to the target nucleicacid. A non-complementary sequence may also be included, such as atarget capture sequence (generally a homopolymer tract, such as apoly-A, poly-T or poly-U tail), promotor sequence, a binding site forRNA transcription, a restriction endonuclease recognition site, orsequences which will confer a desired secondary or tertiary structure,such as a catalytic active site or a hairpin structure, which can beused to facilitate detection and/or amplification. Probes of a definedsequence may be produced by techniques known to those of ordinary skillin the art, such as by chemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5° C. below the melting temperature of the nucleicacid duplex, and even more preferably at least 10° C. below the meltingtemperature of the reaction mixture.

By “substantially homologous,” “substantially corresponding,” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 80% homologous, preferably at least 90% homologous, andmost preferably 100% homologous to an at least 10 contiguous base regionpresent in a reference base sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of homology to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of similarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences that may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of homology between two sequences canalso be expressed in terms of the number of base mismatches present ineach set of at least 10 contiguous bases being compared, which may rangefrom 0 to 2 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 80% complementary, preferably at least 90%complementary, and most preferably 100% complementary to an at least 10contiguous base region present in a target nucleic acid sequence(excluding RNA and DNA equivalents). (Those skilled in the art willreadily appreciate modifications that could be made to the hybridizationassay conditions at various percentages of complementarity to permithybridization of the oligonucleotide to the target sequence whilepreventing unacceptable levels of non-specific hybridization.) Thedegree of complementarity is determined by comparing the order ofnucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of complementaritybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0 to 2 base mismatches.

By “about” is meant the nearest rounded whole number when referring to apercentage of complementarity or homology (e.g., a lower limit of 24.4bases would be 24 bases and a lower limit of 24.5 bases would be 25bases).

By “RNA and DNA equivalents” is meant RNA and DNA molecules having thesame complementary base pair hybridization properties. RNA and DNAequivalents have different sugar moieties (i.e., ribose versusdeoxyribose) and may differ by the presence of uracil in RNA and thyminein DNA. The differences between RNA and DNA equivalents do notcontribute to differences in homology because the equivalents have thesame degree of complementarity to a particular sequence.

By “hybridization” or “hybridize” is meant the ability of two completelyor partially complementary nucleic acid strands to come together underspecified hybridization assay conditions in a parallel or preferablyantiparallel orientation to form a stable structure having adouble-stranded region. The two constituent strands of thisdouble-stranded structure, sometimes called a hybrid, are held togetherby hydrogen bonds. Although these hydrogen bonds most commonly formbetween nucleotides containing the bases adenine and thymine or uracil(A and T or U) or cytosine and guanine (C and G) on single nucleic acidstrands, base pairing can also form between bases which are not membersof these “canonical” pairs. Non-canonical base pairing is well-known inthe art. (See, e.g., ROGER L. P. ADAMS ET AL., THE BIOCHEMISTRY OF THENUCLEIC ACIDS (11^(th) ed. 1992).)

By “preferentially hybridize” is meant that under stringenthybridization conditions, detection probes can hybridize to their targetnucleic acids to form stable probe:target hybrids indicating thepresence of at least one organism of interest, and there is not formed asufficient number of stable probe:non-target hybrids to indicate thepresence of non-targeted organisms, especially phylogenetically closelyrelated organisms. Thus, the probe hybridizes to target nucleic acid toa sufficiently greater extent than to non-target nucleic acid to enableone having ordinary skill in the art to accurately detect the presence(or absence) of nucleic acid derived from T. vaginalis, as appropriate,and distinguish its presence from that of a phylogenetically closelyrelated organism in a test sample. In general, reducing the degree ofcomplementarity between an oligonucleotide sequence and its targetsequence will decrease the degree or rate of hybridization of theoligonucleotide to its target region. However, the inclusion of one ormore non-complementary nucleosides or nucleobases may facilitate theability of an oligonucleotide to discriminate against non-targetorganisms.

Preferential hybridization can be measured using techniques known in theart and described herein, such as in the examples provided below.Preferably, there is at least a 10-fold difference between target andnon-target hybridization signals in a test sample, more preferably atleast a 100-fold difference, and most preferably at least a 1,000-folddifference. Preferably, non-target hybridization signals in a testsample are no more than the background signal level.

By “stringent hybridization conditions,” or “stringent conditions” ismeant conditions permitting a detection probe to preferentiallyhybridize to a target nucleic acid (preferably rRNA or rDNA derived fromT. vaginalis) and not to nucleic acid derived from a closely relatednon-target microorganism. Stringent hybridization conditions may varydepending upon factors including the GC content and length of the probe,the degree of similarity between the probe sequence and sequences ofnon-target sequences which may be present in the test sample, and thetarget sequence. Hybridization conditions include the temperature andthe composition of the hybridization reagents or solutions. Preferredhybridization assay conditions for detecting target nucleic acidsderived from T. vaginalis with the probes of the present inventioncorrespond to a temperature of about 60° C. when the salt concentrationis in the range of about 0.6-0.9 M. Specific hybridization assayconditions are set forth infra in the Examples section and in thesection entitled “Detection Probes to Trichomonas vaginalis RibosomalNucleic Acid.” Other acceptable stringent hybridization conditions couldbe easily ascertained by someone having ordinary skill in the art.

By “assay conditions” is meant conditions permitting stablehybridization of an oligonucleotide to a target nucleic acid. Assayconditions do not require preferential hybridization of theoligonucleotide to the target nucleic acid.

By “consists essentially of” or “consisting essentially of,” when usedwith reference to an oligonucleotide herein, is meant that theoligonucleotide has a base sequence substantially homologous to aspecified base sequence and may have up to four additional bases and/ortwo bases deleted therefrom. Thus, these phrases contain both a sequencelength limitation and a sequence variation limitation. Any additions ordeletions are non-material variations of the specified base sequencewhich do not prevent the oligonucleotide from having its claimedproperty, such as being able to preferentially hybridize under stringenthybridization conditions to its target nucleic acid over non-targetnucleic acids. The oligonucleotide may contain a base sequencesubstantially similar to a specified nucleic acid sequence without anyadditions or deletions. However, a probe or primer containing anoligonucleotide consisting essentially of (or which consists essentiallyof) a specified base sequence may include other nucleic acid moleculeswhich do not participate in hybridization of the probe to the targetnucleic acid and which do not affect such hybridization.

By “nucleic acid duplex,” “duplex,” “nucleic acid hybrid” or “hybrid” ismeant a stable nucleic acid structure comprising a double-stranded,hydrogen-bonded region. Such hybrids include RNA:RNA, RNA:DNA andDNA:DNA duplex molecules and analogs thereof. The structure issufficiently stable to be detectable by any known means, including meansthat do not require a probe associated label. For instance, thedetection method may include a probe-coated substrate that is opticallyactive and sensitive to changes in mass at its surface. Mass changesresult in different reflective and transmissive properties of theoptically active substrate in response to light and serve to indicatethe presence or amount of immobilized target nucleic acid. (Thisexemplary form of optical detection is disclosed by Nygren et al.,“Devices and Methods for Optical Detection of Nucleic AcidHybridization,” U.S. Pat. No. 6,060,237.) Other means for detecting theformation of a nucleic acid duplex that do not require the use of alabeled probe include the use of binding agents, which includeintercalating agents such as ethidium bromide. See, e.g., Higuchi,“Homogenous Methods for Nucleic Amplification and Detection,” U.S. Pat.No. 5,994,056.

By “amplification oligonucleotide” or “primer” is meant anoligonucleotide capable of hybridizing to a target nucleic acid andacting as a primer and/or a promoter template (e.g., for synthesis of acomplementary strand, thereby forming a functional promoter sequence)for the initiation of nucleic acid synthesis. If the amplificationoligonucleotide is designed to initiate RNA synthesis, the primer maycontain a base sequence which is non-complementary to the targetsequence but which is recognized by a RNA polymerase such as a T7, T3,or SP6 RNA polymerase. An amplification oligonucleotide may contain a 3′terminus that is modified to prevent or lessen the rate or amount ofprimer extension. (McDonough et al., “Methods of Amplifying NucleicAcids Using Promoter-Containing Primer Sequences,” U.S. Pat. No.5,766,849, disclose primers and promoter-primers having modified orblocked 3′-ends.) While the amplification oligonucleotides of thepresent invention may be chemically synthesized or derived from avector, they are not naturally occurring nucleic acid molecules.

By “nucleic acid amplification” or “target amplification” is meantincreasing the number of nucleic acid molecules having at least onetarget nucleic acid sequence. Target amplification according to thepresent invention may be either linear or exponential, althoughexponential amplification is preferred.

By “amplification conditions” is meant conditions permitting nucleicacid amplification. Acceptable amplification conditions could be readilyascertained without the exercise of anything more than routineexperimentation by someone having ordinary skill in the art depending onthe particular method of amplification employed.

By “antisense,” “opposite sense,” or “negative sense” is meant a nucleicacid molecule perfectly complementary to a reference, or sense, nucleicacid molecule.

By “sense,” “same-sense,” or “positive sense” is meant a nucleic acidmolecule perfectly homologous to a reference nucleic acid molecule.

By “amplicon” is meant a nucleic acid molecule generated in a nucleicacid amplification reaction and which is derived from a target nucleicacid. An amplicon contains a target nucleic acid sequence that may be ofthe same or opposite sense as the target nucleic acid.

By “derived” is meant that the referred to nucleic acid is obtaineddirectly from an organism or is the product of a nucleic acidamplification. Thus, a nucleic acid that is “derived” from an organismmay be, for example, an antisense RNA molecule which does not naturallyexist in the organism.

By “capture probe” is meant an oligonucleotide that is capable ofbinding to a target nucleic acid (preferably in a region other than thattargeted by a detection probe) and, either directly or indirectly, to asolid support, thereby providing means for immobilizing and isolatingthe target nucleic acid in a test sample. The capture probe includes atarget binding region that hybridizes to the target nucleic acid.Although the capture probe may include a member of ligand-ligate bindingpair (e.g., avidin-biotin linkage) for immobilizing the capture probe ona solid support, preferred capture probes include an immobilized probebinding region that hybridizes to an immobilized probe bound to a solidsupport. While the capture probe preferably hybridizes to both thetarget nucleic acid and the immobilized probe under stringentconditions, the target binding and the immobilized probe binding regionsof the capture probe may be designed to bind to their target sequencesunder different hybridization conditions. In this way, the capture probemay be designed so that it first hybridizes to the target nucleic acidunder more favorable in solution kinetics before adjusting theconditions to permit hybridization of the immobilized probe bindingregion to the immobilized probe. The target binding and immobilizedprobe binding regions may be contained within the same oligonucleotide,directly adjoining each other or separated by one or more optionallymodified nucleotides, or these regions may be joined to each other bymeans of a non-nucleotide linker.

By “target binding region” is meant that portion of an oligonucleotidewhich stably binds to a target sequence present in a target nucleicacid, a DNA or RNA equivalent of the target sequence or a complement ofthe target sequence under assay conditions. The assay conditions may bestringent hybridization conditions or amplification conditions.

By “immobilized probe binding region” is meant that portion of anoligonucleotide which hybridizes to an immobilized probe under assayconditions.

By “homopolymer tail” in the claims is meant a contiguous base sequenceof at least 10 identical bases (e.g., 10 contiguous adenines orthymines).

By “immobilized probe” is meant an oligonucleotide for joining a captureprobe to an immobilized support. The immobilized probe is joined eitherdirectly or indirectly to the solid support by a linkage or interactionwhich remains stable under the conditions employed to hybridize thecapture probe to the target nucleic acid and to the immobilized probe,whether those conditions are the same or different. The immobilizedprobe facilitates separation of the bound target nucleic acid fromunbound materials in a sample.

By “isolate” or “isolating” is meant that at least a portion of thetarget nucleic acid present in a test sample is concentrated within areaction receptacle or on a reaction device or solid carrier (e.g., testtube, cuvette, microtiter plate well, nitrocellulose filter, slide orpipette tip) in a fixed or releasable manner so that the target nucleicacid can be purified without significant loss of the target nucleic acidfrom the receptacle, device or carrier.

By “purify” or “purifying” is meant that one or more components of thetest sample are removed from one or more other components of the sample.Sample components to be purified may include viruses, nucleic acids or,in particular, target nucleic acids in a generally aqueous solutionphase which may also include undesirable materials such as proteins,carbohydrates, lipids, non-target nucleic acid and/or labeled probes.Preferably, the purifying step removes at least about 70%, morepreferably at least about 90% and, even more preferably, at least about95% of the undesirable components present in the sample.

By “helper probe” or “helper oligonucleotide” is meant anoligonucleotide designed to hybridize to a target nucleic acid at adifferent locus than that of a detection probe, thereby eitherincreasing the rate of hybridization of the probe to the target nucleicacid, increasing the melting temperature (T_(m)) of the probe:targethybrid, or both.

By “phylogenetically closely related” is meant that the organisms areclosely related to each other in an evolutionary sense and thereforewould be expected to have a higher total nucleic acid sequence homologythan organisms that are more distantly related. Organisms occupyingadjacent and next to adjacent positions on the phylogenetic tree areclosely related. Organisms occupying positions farther away thanadjacent or next to adjacent positions on the phylogenetic tree willstill be closely related if they have significant total nucleic acidsequence homology.

B. Hybridization Conditions and Probe Design

Hybridization reaction conditions, most importantly the temperature ofhybridization and the concentration of salt in the hybridizationsolution, can be selected to allow the detection probes or, in somecases, amplification oligonucleotides of the present invention topreferentially hybridize to a T. vaginalis-derived target nucleic acidand not to other non-target nucleic acids suspected of being present ina test sample. At decreased salt concentrations and/or increasedtemperatures (conditions of increased stringency) the extent of nucleicacid hybridization decreases as hydrogen bonding between pairednucleobases in the double-stranded hybrid molecule is disrupted. Thisprocess is known as “melting.”

Generally speaking, the most stable hybrids are those having the largestnumber of contiguous, perfectly matched (i.e., hydrogen-bonded)nucleotide base pairs. Such hybrids would usually be expected to be thelast to melt as the stringency of the hybridization conditionsincreases. However, a double-stranded nucleic acid region containing oneor more mismatched, “non-canonical,” or imperfect base pairs (resultingin weaker or non-existent base pairing at that position in thenucleotide sequence of a nucleic acid) may still be sufficiently stableunder conditions of relatively high stringency to allow the nucleic acidhybrid to be formed and detected in a hybridization assay withoutcross-reacting with other, non-selected nucleic acids which may bepresent in a test sample.

Hence, depending on the degree of similarity between the nucleotidesequences of the target nucleic acid and those of non-target nucleicacids belonging to phylogenetically distinct, but closely-relatedorganisms on one hand, and the degree of complementarity between thenucleotide sequences of a particular detection probe or amplificationoligonucleotide and those of the target and non-target nucleic acids onthe other, one or more mismatches will not necessarily defeat theability of an oligonucleotide contained in the probe or amplificationoligonucleotide to hybridize to the target nucleic acid and not tonon-target nucleic acids.

The detection probes of the present invention were chosen, selected,and/or designed to maximize the difference between the meltingtemperatures of the probe:target hybrid (T_(m), defined as thetemperature at which half of the potentially double-stranded moleculesin a given reaction mixture are in a single-stranded, denatured state)and the T_(m) of a mismatched hybrid formed between the probe andribosomal RNA (rRNA) or ribosomal DNA (rDNA) of the phylogeneticallymost closely-related organisms expected to be present in the testsample, but not sought to be detected. While the unlabeled amplificationoligonucleotides, capture probes and helper probes need not have such anextremely high degree of specificity as the detection probe to be usefulin the present invention, they are designed in a similar manner topreferentially hybridize to one or more target nucleic acids over othernucleic acids under specified amplification, assay or stringenthybridization conditions.

To facilitate the identification of nucleic acid sequences to be used inthe design of probes, nucleotide sequences from different organisms werefirst aligned to maximize homology. The source organisms and theassociated nucleotide sequences used for this comparison were obtainedfrom the GenBank database and had the following accession numbers:Trichomonas vaginalis(Accession No. U17510), Trimastix pyriformis(Accession No. AF244903), Dientamoeba fragilis (Accession No. U37461),Trichomonas gallinae (Accession No. U86614), Trichomonas tenax(AccessionNos. D49495 and U37711), Tetratrichomonas gallinarum (Accession No.AF124608), Kalotermes flavicollis (Accession No. AF215856), Trichomitustrypanoides (Accession No. X79559), Hodotermopsis sjoestedti (AccessionNo. AB032234), Pentatrichomonas hominis (Accession No. AF124609),Pseudotrypanosoma giganteum (Accession No. AF052706), Ditrichomonashonigbergi (Accession No. U17505), Monotrichomonas species ATCC50693(Accession No. AF072905), Pseudotrichomonas keilini (Accession No.U17511), Monocercomonas species ATCC 50210 (Accession No. U17507),Tritrichomonas foetus (Accession No. U17509) and Entamoeba histolytica(Accession No. X64142).

Within the rRNA molecule there is a close relationship between secondarystructure (caused in part by intra-molecular hydrogen bonding) andfunction. This fact imposes restrictions on evolutionary changes in theprimary nucleotide sequence causing the secondary structure to bemaintained. For example, if a base is changed in one “strand” of adouble helix (due to intra-molecular hydrogen bonding, both “strands”are part of the same rRNA molecule), a compensating substitution usuallyoccurs in the primary sequence of the other “strand” in order topreserve complementarity (this is referred to as co-variance), and thusthe necessary secondary structure. This allows two very different rRNAsequences to be aligned based both on the conserved primary sequence andalso on the conserved secondary structure elements. Potential targetsequences for the detection probes described herein were identified bynoting variations in the homology of the aligned sequences.

The sequence evolution at each of the variable regions is mostlydivergent. Because of the divergence, corresponding rRNA variableregions of more distant phylogenetic relatives of T. vaginalis showgreater differences from T. vaginalis rRNA than do the rRNAs ofphylogenetically closer relatives. Sufficient variation between T.vaginalis and other organisms was observed to identify preferred targetsites and to design detection probes useful for distinguishing T.vaginalis over non-T. vaginalis organisms in a test sample, particularlyTrichomonas tenax, the most closely related organism to T. vaginalis.

Merely identifying putatively unique potential target nucleotidesequences does not guarantee that a functionally species-specificdetection probe may be made to hybridize to T. vaginalis rRNA or rDNAcomprising that sequence. Various other factors will determine thesuitability of a nucleic acid locus as a target site for genus-specificor species-specific probes. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particularoligonucleotide, whether perfectly complementary to its target or not.The importance and effect of various assay conditions are known to thoseskilled in the art and are disclosed by Hogan et al., “Nucleic AcidProbes for Detection and/or Quantitation of Non-Viral Organisms,” U.S.Pat. No. 5,840,488; Hogan et al., “Nucleic Acid Probes to Mycobacteriumgordonae,” U.S. Pat. No. 5,216,143; and Kohne, “Method for Detection,Identification and Quantitation of Non-Viral Organisms,” U.S. Pat. No.4,851,330. The contents of each of the foregoing references is herebyincorporated by reference herein.

The desired temperature of hybridization and the hybridization solutioncomposition (such as salt concentration, detergents, and other solutes)can also greatly affect the stability of double-stranded hybrids.Conditions such as ionic strength and the temperature at which a probewill be allowed to hybridize to a target must be taken into account inconstructing a genus-specific or species-specific probe. The thermalstability of hybrid nucleic acids generally increases with the ionicstrength of the reaction mixture. On the other hand, chemical reagentsthat disrupt hydrogen bonds, such as formamide, urea, dimethyl sulfoxideand alcohols, can greatly reduce the thermal stability of the hybrids.

To maximize the specificity of a probe for its target, the subjectprobes of the present invention were designed to hybridize to theirtargets under conditions of high stringency. Under such conditions onlysingle nucleic acid strands having a high degree of complementarity willhybridize to each other. Single nucleic acid strands without such a highdegree of complementarity will not form hybrids. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity that should exist between two nucleic acid strands inorder to form a hybrid. Stringency is chosen to maximize the differencein stability between the hybrid formed between the probe and the targetnucleic acid and potential hybrids between the probe and any non-targetnucleic acids present in a test sample.

Proper specificity may be achieved by minimizing the length of thedetection probe having perfect complementarity to sequences ofnon-target organisms, by avoiding G and C rich regions ofcomplementarity to non-target nucleic acids, and by constructing theprobe to contain as many destabilizing mismatches to non-targetsequences as possible. Whether a probe is appropriate for detecting onlya specific type of organism depends largely on the thermal stabilitydifference between probe:target hybrids versus probe:non-target hybrids.In designing probes, the differences in these T_(m) values should be aslarge as possible (preferably 2-5° C. or more). Manipulation of theT_(m) can be accomplished by changes to probe length and probecomposition (e.g., GC content versus AT content).

In general, the optimal hybridization temperature for oligonucleotideprobes is approximately 5° C. below the melting temperature for a givenduplex. Incubation at temperatures below the optimum temperature mayallow mismatched base sequences to hybridize and can therefore decreasespecificity. The longer the probe, the more hydrogen bonding betweenbase pairs and, in general, the higher the T_(m). Increasing thepercentage of G and C also increases the T_(m) because G-C base pairsexhibit additional hydrogen bonding and therefore greater thermalstability than A-T base pairs. Such considerations are known in the art.(See, e.g., J. SAMBROOKET AL., MOLECULAR CLONING: A LABORATORY MANUAL,ch. 11 (2^(nd) ed. 1989).)

A preferred method to determine T_(m) measures hybridization using thewell known Hybridization Protection Assay (HPA) disclosed by Arnold etal., “Homogenous Protection Assay,” U.S. Pat. No. 5,283,174, thecontents of which are hereby incorporated by reference herein. The T_(m)can be measured using HPA in the following manner. Probe molecules arelabeled with an acridinium ester and permitted to form probe:targethybrids in a lithium succinate buffer (0.1 M lithium succinate buffer,pH 4.7, 20 mM EDTA, 15 mM aldrithiol-2, 1.2 M LiCl, 3% (v/v) ethanolabsolute, 2% (w/v) lithium lauryl sulfate) using an excess amount oftarget. Aliquots of the solution containing the probe:target hybrids arethen diluted in the lithium succinate buffered solution and incubatedfor five minutes at various temperatures starting below that of theanticipated T_(m) (typically 55° C.) and increasing in 2-5° C.increments. This solution is then diluted with a mild alkaline boratebuffer (600 mM boric acid, 240 mM NaOH, 1% (v/v) TRITON® X-100detergent, pH 8.5) and incubated at an equal or lower temperature (forexample 50° C.) for ten minutes.

Under these conditions the acridinium ester attached to thesingle-stranded probe is hydrolyzed, while the acridinium ester attachedto hybridized probe is relatively protected from hydrolysis. Thus, theamount of acridinium ester remaining after hydrolysis treatment isproportional to the number of hybrid molecules. The remaining acridiniumester can be measured by monitoring the chemiluminescence produced fromthe remaining acridinium ester by adding hydrogen peroxide and alkali tothe solution. Chemiluminescence can be measured in a luminometer, suchas a LEADER® HC+ Luminometer (Gen-Probe Incorporated; San Diego, Calif.;Cat. No. 4747). The resulting data is plotted as percent of maximumsignal (usually from the lowest temperature) versus temperature. TheT_(m) is defined as the temperature at which 50% of the maximum signalremains. In addition to the method above, T_(m) may be determined byisotopic methods known to those skilled in the art (see, e.g., Hogan etal., U.S. Pat. No. 5,840,488).

To ensure specificity of a detection probe for its target, it ispreferable to design probes that hybridize only to target nucleic acidunder conditions of high stringency. Only highly complementary sequenceswill form hybrids under conditions of high stringency. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two sequences in order for a stablehybrid to form. Stringency should be chosen to maximize the differencein stability between the probe:target hybrid and potentialprobe:non-target hybrids.

Examples of specific stringent hybridization conditions are provided inthe Examples section infra. Of course, alternative stringenthybridization conditions can be determined by those of ordinary skill inthe art based on the present disclosure. (See, e.g., SAMBROOK ET AL.,supra, ch. 11.)

The length of the target nucleic acid sequence region and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which may be used to design probes with the desiredhybridization characteristics. In other cases, one probe may besignificantly better with regard to specificity than another thatdiffers from it merely by a single base. While it is possible fornucleic acids that are not perfectly complementary to hybridize, thelongest stretch of perfectly complementary bases, as well as the basecompositions, will generally determine hybrid stability.

Regions of rRNA known to form strong internal structures inhibitory tohybridization are less preferred target regions, especially in assayswhere helper probes described infra are not used. Likewise, probes withextensive self-complementarity are generally to be avoided, withspecific exceptions being discussed below. If a strand is wholly orpartially involved in an intramolecular or intermolecular hybrid, itwill be less able to participate in the formation of a newintermolecular probe:target hybrid without a change in the reactionconditions. Ribosomal RNA molecules are known to form very stableintramolecular helices and secondary structures by hydrogen bonding. Bydesigning a probe to a region of the target nucleic acid which remainssubstantially single-stranded under hybridization conditions, the rateand extent of hybridization between probe and target may be increased.

A genomic ribosomal nucleic acid (rDNA) target occurs naturally in adouble-stranded form, as does the product of the polymerase chainreaction (PCR). These double-stranded targets are naturally inhibitoryto hybridization with a probe and require denaturation prior tohybridization. Appropriate denaturation and hybridization conditions areknown in the art (see, e.g., Southern, E. M., J. Mol. Biol., 98:503(1975)).

A number of formulae are available which will provide an estimate of themelting temperature for perfectly matched oligonucleotides to theirtarget nucleic acids. One such formula is the following:

T _(m)=81.5+16.6(log₁₀[Na+])+0.41(fraction G+C)−(600/N)

(where N=the length of the oligonucleotide in number of nucleotides)provides a good estimate of the T_(m) for oligonucleotides between 14and 60 to 70 nucleotides in length. From such calculations, subsequentempirical verification or “fine tuning” of the T_(m) may be made usingscreening techniques well known in the art. For further information onhybridization and oligonucleotide probes reference may be made toSAMBROOK ET AL., supra, ch. 11. This reference, among others well knownin the art, also provides estimates of the effect of mismatches on theT_(m) of a hybrid. Thus, from the known nucleotide sequence of a givenregion of the ribosomal RNA (or rDNA) of two or more organisms,oligonucleotides may be designed which will distinguish these organismsfrom one another.

C. Nucleic Acid Amplification

Preferably, the amplification oligonucleotides of the present inventionare oligodeoxynucleotides and are sufficiently long to be used as asubstrate for the synthesis of extension products by a nucleic acidpolymerase. Optimal amplification oligonucleotide length should takeinto account several factors, including the temperature of reaction, thestructure and base composition of the amplification oligonucleotide, andhow the amplification oligonucleotide is to be used. For example, foroptimal specificity the oligonucleotide amplification oligonucleotidegenerally should be at least 12 bases in length, depending on thecomplexity of the target nucleic acid sequence. If such specificity isnot essential, shorter amplification oligonucleotides may be used. Insuch a case, it may be desirable to carry out the reaction at coolertemperatures in order to form stable hybrid complexes with the templatenucleic acid.

Useful guidelines for designing amplification oligonucleotides anddetection probes with desired characteristics are described infra in thesection entitled “Preparation of Oligonucleotides.” Optimal sites foramplifying and probing contain at least two, and preferably three,conserved regions of T. vaginalis nucleic acid. These regions are about15 to 350 bases in length, and preferably between about 15 and 150 basesin length.

The degree of amplification observed with a set of amplificationoligonucleotides (e.g., primers and/or promoter-primers) depends onseveral factors, including the ability of the amplificationoligonucleotides to hybridize to their specific target sequences andtheir ability to be extended or copied enzymatically. Whileamplification oligonucleotides of different lengths and basecompositions may be used, amplification oligonucleotides preferred inthis invention have target binding regions of 18 to 40 bases with apredicted T_(m) to target of about 42° C.

Parameters affecting probe hybridization, such as T_(m),complementarity, and secondary structure of the target sequence, alsoaffect amplification oligonucleotide hybridization and thereforeperformance of the amplification oligonucleotides. The degree ofnon-specific extension (primer-dimer or non-target copying) can alsoaffect amplification efficiency. Thus, amplification oligonucleotidesare selected to have low self-complementarity or cross-complementarity,particularly at the 3′ ends of their sequences. Notwithstanding, itshould be noted that the “signal primers” described infra could bemodified to include regions of self-complementarity, therebytransforming them into “molecular torch” or “molecular beacon” signalprimers, such as these terms are defined below. Lengthy homopolymer runsand high GC content are avoided to reduce spurious primer extension.Computer programs are available to aid in this aspect of the design,including Oligo Tech analysis software which is available from OligosEtc. Inc. and can be accessed on the World Wide Web at the followingURL: http://www.oligosetc.com.

A nucleic acid polymerase used in conjunction with the amplificationoligonucleotides of the present invention refers to a chemical,physical, or biological agent that incorporates either ribonucleotidesor deoxyribonucleotides, or both, into a nucleic acid polymer, orstrand, in a template-dependent manner. Examples of nucleic acidpolymerases include DNA-directed DNA polymerases, RNA-directed DNApolymerases, and RNA-directed RNA polymerases. DNA polymerases bringabout nucleic acid synthesis in a template-dependent manner and in a 5′to 3′ direction. Because of the typical anti-parallel orientation of thetwo strands in a double-stranded nucleic acid, this direction is from a3′ region on the template to a 5′ region on the template. Examples ofDNA-directed DNA polymerases include E. coli DNA polymerase I, thethermostable DNA polymerase from Thermus aquaticus (Taq), and the largefragment of DNA polymerase I from Bacillus stearothermophilis (Bst).Examples of RNA directed DNA polymerases include various retroviralreverse transcriptases, such as Moloney murine leukemia virus (MMLV)reverse transcriptase or avian myeloblastosis virus (AMV) reversetranscriptase.

During most nucleic acid amplification reactions, a nucleic acidpolymerase adds nucleotide residues to the 3′ end of the primer usingthe target nucleic acid as a template, thus synthesizing a secondnucleic acid strand having a nucleotide sequence partially or completelycomplementary to a region of the target nucleic acid. In many nucleicacid amplification reactions, the two strands comprising the resultingdouble-stranded structure must be separated by chemical or physicalmeans in order to allow the amplification reaction to proceed.Alternatively, the newly synthesized template strand may be madeavailable for hybridization with a second primer or promoter-primer byother means, such as through strand displacement or the use of anucleolytic enzyme which digests part or all of the original targetstrand. In this way the process may be repeated through a number ofcycles, resulting in a large increase in the number of nucleic acidmolecules having the target nucleotide sequence.

Either the first or second amplification oligonucleotide, or both, maybe a promoter-primer. (In some applications, the amplificationoligonucleotides may only consist of promoter-primers which arecomplementary to the sense strand, as disclosed by Kacian et al.,“Nucleic Acid Sequence Amplification Method, Composition and Kit,” U.S.Pat. No. 5,554,516.) A promoter-primer usually contains anoligonucleotide that is not complementary to a nucleotide sequencepresent in the target nucleic acid molecule or primer extensionproduct(s) (see Kacian et al., “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,399,491, for a description of sucholigonucleotides). These non-complementary sequences may be located 5′to the complementary sequences on the amplification oligonucleotide andmay provide a locus for initiation of RNA synthesis when madedouble-stranded through the action of a nucleic acid polymerase. Thepromoter thus provided may allow for the in vitro transcription ofmultiple RNA copies of the target nucleic acid sequence. It will beappreciated that when reference is made to a primer in thisspecification, such reference is intended to include the primer aspectof a promoter-primer as well, unless the context of the referenceclearly indicates otherwise.

In some amplification systems (see, e.g., the amplification methodsdisclosed by Dattagupta et al., “Isothermal Strand DisplacementAmplification,” U.S. Pat. No. 6,087,133), the amplificationoligonucleotides may contain 5′ non-complementary nucleotides whichassist in strand displacement. Furthermore, when used in conjunctionwith a nucleic acid polymerase having 5′ exonuclease activity, theamplification oligonucleotides may have modifications at their 5′ end toprevent enzymatic digestion. Alternatively, the nucleic acid polymerasemay be modified to remove the 5′ exonuclease activity, such as bytreatment with a protease that generates an active polymerase fragmentwith no such nuclease activity. In such a case the primers need not bemodified at their 5′ ends.

1. Preparation of Oligonucleotides

The detection probes, capture probes, helper probes and amplificationoligonucleotides of the present invention can be readily prepared bymethods known in the art. Preferably, the oligonucleotides aresynthesized using solid phase methods. For example, Caruthers describesusing standard phosphoramidite solid-phase chemistry to join nucleotidesby phosphodiester linkages. See Caruthers et al., “Chemical Synthesis ofDeoxynucleotides by the Phosphoramidite Method,” Methods Enzymol.,154:287 (1987). Automated solid-phase chemical synthesis usingcyanoethyl phosphoramidite precursors has been described by Barone. SeeBarone et al., “In Situ Activation of bis-dialkylaminephosphines—a NewMethod for Synthesizing Deoxyoligonucleotides on Polymer Supports,”Nucleic Acids Res., 12(10):4051 (1984). Likewise, Batt, “Method andReagent for Sulfurization of Organophosphorous Compounds,” U.S. Pat. No.5,449,769, discloses a procedure for synthesizing oligonucleotidescontaining phosphorothioate linkages. In addition, Riley et al.,“Process for the Purification of Oligomers,” U.S. Pat. No. 5,811,538disclose the synthesis of oligonucleotides having different linkages,including methylphosphonate linkages. Moreover, methods for the organicsynthesis of oligonucleotides are known to those of skill in the art andare described in, for example, SAMBROOK ET AL., supra, ch. 10.

Following synthesis of a particular oligonucleotide, several differentprocedures may be utilized to purify and control the quality of theoligonucleotide. Suitable procedures include polyacrylamide gelelectrophoresis or high pressure liquid chromatography. Both of theseprocedures are well known to those skilled in the art.

All of the oligonucleotides of the present invention, whether detectionprobes, helper probes, capture probes or amplification oligonucleotides,may be modified with chemical groups to enhance their performance or tofacilitate the characterization of amplification products.

For example, backbone-modified oligonucleotides such as those havingphosphorothioate, methylphosphonate, 2′-O-alkyl, or peptide groups whichrender the oligonucleotides resistant to the nucleolytic activity ofcertain polymerases or to nuclease enzymes may allow the use of suchenzymes in an amplification or other reaction. Another example of amodification involves using non-nucleotide linkers incorporated betweennucleotides in the nucleic acid chain of a probe or primer, and which donot prevent hybridization of a probe or hybridization and elongation ofa primer. (See Arnold et al., “Non-Nucleotide Linking Reagents forNucleotide Probes,” U.S. Pat. No. 6,031,091, the contents of which arehereby incorporated by reference herein.) The oligonucleotides of thepresent invention may also contain mixtures of the desired modified andnatural nucleotides.

The 3′ end of an amplification oligonucleotide, particularly apromoter-primer, may be modified or blocked to prevent or inhibitinitiation of DNA synthesis, as disclosed by Kacian et al., U.S. Pat.No. 5,554,516. The 3′ end of the primer can be modified in a variety ofways well known in the art. By way of example, appropriate modificationsto a promoter-primer can include the addition of ribonucleotides, 3′deoxynucleotide residues (e.g., cordycepin), 2′,3′-dideoxynucleotideresidues, modified nucleotides such as phosphorothioates, andnon-nucleotide linkages such as those disclosed by Arnold et al. in U.S.Pat. No. 6,031,091 or alkane-diol modifications (see Wilk et al.,“Backbone-Modified Oligonucleotides Containing a Butanediol-1,3 Moietyas a ‘Vicarious Segment’ for the Deoxyribosyl Moiety—Synthesis andEnzyme Studies,” Nucleic Acids Res., 18(8):2065 (1990)), or themodification may simply consist of a region 3′ to the priming sequencethat is non-complementary to the target nucleic acid sequence.Additionally, a mixture of different 3′ blocked promoter-primers or of3′ blocked and unblocked promoter-primers may increase the efficiency ofnucleic acid amplification, as described therein.

As disclosed above, the 5′ end of primers may be modified to beresistant to the 5′-exonuclease activity present in some nucleic acidpolymerases. Such modifications can be carried out by adding anon-nucleotide group to the terminal 5′ nucleotide of the primer usingtechniques such as those disclosed by Arnold et al., U.S. Pat. No.6,031,091.

Once synthesized, a selected oligonucleotide may be labeled by any wellknown method (see, e.g., SAMBROOK ET AL., supra, ch. 10). Useful labelsinclude radioisotopes as well as non-radioactive reporting groups.Isotopic labels include ³H, ³⁵S, ³²P, ¹²⁵I, ⁵⁷Co, and ¹⁴C. Isotopiclabels can be introduced into the oligonucleotide by techniques known inthe art such as nick translation, end labeling, second strand synthesis,the use of reverse transcription, and by chemical methods. When usingradiolabeled probes, hybridization can be detected by autoradiography,scintillation counting, or gamma counting. The detection method selectedwill depend upon the particular radioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups as disclosed by Arnold et al.,U.S. Pat. No. 6,031,091. Non-isotopic labels include fluorescentmolecules (individual labels or combinations of labels, such as thefluorescence resonance energy transfer (FRET) pairs disclosed by Tyagiet al., “Detectably Labeled Dual Conformation Oligonucleotide Probes,”U.S. Pat. No. 5,925,517), chemiluminescent molecules, enzymes,cofactors, enzyme substrates, haptens, or other ligands.

With the detection probes of the present invention, the probes arepreferably labeled using of a non-nucleotide linker with an acridiniumester. Acridinium ester labeling may be performed as disclosed by Arnoldet al., “Acridinium Ester Labelling and Purification of NucleotideProbes,” U.S. Pat. No. 5,185,439, the contents of which are herebyincorporated by reference herein.

2. Amplification of Trichomonas vaginalis Ribosomal Nucleic Acid

The amplification oligonucleotides of the present invention are directedto 18S regions of ribosomal nucleic acid derived from T. vaginalis.These amplification oligonucleotides may flank, overlap, or be containedwithin at least one of the target sequences of a detection probe (or itscomplement) used to detect the presence of T. vaginalis in a nucleicacid amplification assay. As indicated above, the amplificationoligonucleotides may also include non-complementary bases at their 5′ends comprising a promoter sequence able to bind a RNA polymerase anddirect RNA transcription using the target nucleic acid as a template. AT7 promoter sequence, such as SEQ ID NO:89, may be used.

Amplification oligonucleotides of the present invention are capable ofamplifying a target region of nucleic acid derived from T. vaginalisunder amplification conditions. The amplification oligonucleotides havea target binding region up to 40 bases in length which stably hybridizesto a target sequence selected from the group consisting of SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59,SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64under amplification conditions. The amplification oligonucleotide doesnot include any other base sequences which stably hybridize to nucleicacid derived from T. vaginalis under amplification conditions.Preferably, the base sequence of the target binding region comprises,overlaps with, consists essentially of, consists of, substantiallycorresponds to, or is contained within a base sequence selected from thegroup consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87 and SEQ ID NO:88.

Alternatively, amplification oligonucleotides of the present inventionconsist of a target binding region up to 40 bases in length and anoptional 5′ sequence which is recognized by a RNA polymerase or whichenhances initiation or elongation by a RNA polymerase, where theamplification oligonucleotide will, when contacted with a nucleic acidpolymerase under amplification conditions, bind to or cause extensionthrough a nucleic acid region having a base sequence selected from thegroup consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87 or SEQ ID NO:88.

In one preferred embodiment, a set of at least two amplificationoligonucleotides for amplifying T. vaginalis-derived nucleic acid isprovided which includes: (i) a first amplification oligonucleotidehaving a target binding region up to 40 bases in length which stablyhybridizes to a target sequence selected from the group consisting ofSEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36 underamplification conditions; and (ii) a second amplificationoligonucleotide having a target binding region up to 40 bases in lengthwhich stably hybridizes to a target sequence selected from the groupconsisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40under amplification conditions. Preferably, the first amplificationoligonucleotide has a target binding region which includes a basesequence comprising, overlapping with, consisting essentially of,consisting of, substantially corresponding to, or contained within abase sequence selected from the group consisting of SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47 and SEQ ID NO:48, and the second amplification oligonucleotide hasa target binding region which includes a base sequence comprising,overlapping with, consisting essentially of, consisting of,substantially corresponding to, or contained within a base sequenceselected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 SEQ ID NO:55 and SEQ IDNO:56. More preferably, the base sequence of the target binding regionof the first amplification oligonucleotide comprises, overlaps with,consists essentially of, consists of, substantially corresponds to, oris contained within the base sequence of SEQ ID NO:41 or SEQ ID NO:45,and the base sequence of the target binding region of the secondamplification oligonucleotide comprises, overlaps with, consistsessentially of, consists of, substantially corresponds to, or iscontained within the base sequence of SEQ ID NO:51 or SEQ ID NO:55. Thesecond amplification oligonucleotide preferably includes a 5′ promotersequence (e.g., the T7 promoter sequence of SEQ ID NO:89).

In another preferred embodiment, a set of at least two amplificationoligonucleotides for amplifying T. vaginalis-derived nucleic acid isprovided which includes: (i) a first amplification oligonucleotidehaving a target binding region up to 40 bases in length which stablyhybridizes to a target sequence selected from the group consisting ofSEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60 underamplification conditions; and (ii) a second amplificationoligonucleotide having a target binding region up to 40 bases in lengthwhich stably hybridizes to a target sequence selected from the groupconsisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64under amplification conditions. Preferably, first amplificationoligonucleotide has a target binding region which includes a basesequence comprising, overlapping with, consisting essentially of,consisting of, substantially corresponding to, or contained within abase sequence selected from the group consisting of SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 and SEQ IDNO:76, and the second amplification oligonucleotide has a target bindingregion which includes a base sequence comprising, overlapping with,consisting essentially of, consisting of, substantially correspondingto, or contained within a base sequence of selected from the groupconsisting of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85,SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88. More preferably, the basesequence of the target binding region of the first amplificationoligonucleotide comprises, overlaps with, consists essentially of,consists of, substantially corresponds to, or is contained within thebase sequence of SEQ ID NO:65, SEQ ID NO:69 or SEQ ID NO:73, and basesequence of the target binding region of the second amplificationoligonucleotide comprises, overlaps with, consists essentially of,consists of, substantially corresponds to, or is contained within thebase sequence of SEQ ID NO:79, SEQ ID NO:83 or SEQ ID NO:87. The secondamplification oligonucleotide preferably includes a 5′ promoter sequence(e.g., the T7 promoter sequence of SEQ ID NO:89).

Amplification oligonucleotides of the present invention may havemodifications, such as blocked 3′ and/or 5′ termini (as discussed above)or sequence additions including, but not limited to, a specificnucleotide sequence recognized by a RNA polymerase (e.g., a promotersequence for T7, T3 or SP6 RNA polymerase), a sequence which enhancesinitiation or elongation of RNA transcription by a RNA polymerase, or asequence which may provide for intra-molecular base pairing andencourage the formation of secondary or tertiary nucleic acidstructures.

Amplification oligonucleotides are used in any suitable nucleic acidamplification procedure now known or later developed. Existingamplification procedures include the polymerase chain reaction (PCR),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), self-sustained sequence replication (3SR), ligasechain reaction (LCR), strand displacement amplification (SDA), andLoop-Mediated Isothermal Amplification (LAMP), each of which is wellknown in the art. See, e.g., Mullis, “Process for Amplifying NucleicAcid Sequences,” U.S. Pat. No. 4,683,202; Erlich et al., “Kits forAmplifying and Detecting Nucleic Acid Sequences,” U.S. Pat. No.6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahyet al., “Self-sustained Sequence Replication (3SR): An IsothermalTranscription-Based Amplification System Alternative to PCR,” PCRMethods and Applications, 1:25-33 (1991); Kacian et al., U.S. Pat. No.5,399,491; Kacian et al., “Nucleic Acid Sequence Amplification Methods,”U.S. Pat. No. 5,480,784; Davey et al., “Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,554,517; Birkenmeyer et al., “Amplification ofTarget Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat.No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Usingthe Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al.,“Process for Synthesizing Nucleic Acid,” European Patent Application No.1 020 534 A1; Dattagupta et al., “Isothermal Strand DisplacementAmplification,” U.S. Pat. No. 6,214,587; and HELEN H. LEE ET AL.,NUCLEIC ACID AMPLIFICATION TECHNOLOGIES: APPLICATION TO DISEASEDIAGNOSIS (1997). (Each of the foregoing amplification references ishereby incorporated by reference herein.) Any other amplificationprocedure which meets the definition of “nucleic acid amplification”supra is also contemplated by the inventors.

Amplification oligonucleotides of the present invention are preferablyunlabeled but may include one or more reporter groups to facilitatedetection of a target nucleic acid in combination with or exclusive of adetection probe. A wide variety of methods are available to detect anamplified target sequence. For example, the nucleotide substrates or theamplification oligonucleotides can include a detectable label that isincorporated into newly synthesized DNA. The resulting labeledamplification product is then generally separated from the unusedlabeled nucleotides or amplification oligonucleotides and the label isdetected in the separated product fraction. (See, e.g., Wu, “Detectionof Amplified Nucleic Acid Using Secondary Capture Oligonucleotides andTest Kit,” U.S. Pat. No. 5,387,510.)

A separation step is not required, however, if the amplificationoligonucleotide is modified by, for example, linking it to aninteracting label pair, such as two dyes which form a donor/acceptor dyepair. The modified amplification oligonucleotide can be designed so thatthe fluorescence of one dye pair member remains quenched by the otherdye pair member, so long as the amplification oligonucleotide does nothybridize to target nucleic acid, thereby physically separating the twodyes. Moreover, the amplification oligonucleotide can be furthermodified to include a restriction endonuclease recognition sitepositioned between the two dyes so that when a hybrid is formed betweenthe modified amplification oligonucleotide and target nucleic acid, therestriction endonuclease recognition site is rendered double-strandedand available for cleavage or nicking by an appropriate restrictionendonuclease. Cleavage or nicking of the hybrid then separates the twodyes, resulting in a change in fluorescence due to decreased quenchingwhich can be detected as an indication of the presence of the targetorganism in the test sample. This type of modified amplificationoligonucleotide, referred to as a “signal primer,” is disclosed byNadeau et al., “Detection of Nucleic Acids by Fluorescence Quenching,”U.S. Pat. No. 6,054,279.

Substances which can serve as useful detectable labels are well known inthe art and include radioactive isotopes, fluorescent molecules,chemiluminescent molecules, chromophores, as well as ligands such asbiotin and haptens which, while not directly detectable, can be readilydetected by a reaction with labeled forms of their specific bindingpartners, e.g., avidin and antibodies, respectively.

Another approach is to detect the amplification product by hybridizationwith a detectably labeled oligonucleotide probe and measuring theresulting hybrids in any conventional manner. In particular, the productcan be assayed by hybridizing a chemiluminescent acridiniumester-labeled oligonucleotide probe to the target sequence, selectivelyhydrolyzing the acridinium ester present on unhybridized probe, andmeasuring the chemiluminescence produced from the remaining acridiniumester in a luminometer. (See, e.g., Arnold et al., U.S. Pat. No.5,283,174, and NORMAN C. NELSON ET AL., NONISOTOPIC PROBING, BLOTTING,AND SEQUENCING, ch. 17 (Larry J. Kricka ed., 2d ed. 1995).)

Because genitourinary specimens tend to contain large amounts of T.vaginalis when an individual is infected with the organism, it may bedesirable to include a co-amplifiable pseudo target in the amplificationreaction mixture in order to render the assay less sensitive, especiallywhen quantification is an objective of the assay. Pseudo targets andtheir uses are disclosed by Nunomura, “Polynucleotide AmplificationMethod,” U.S. Pat. No. 6,294,338, the contents of which are herebyincluded by reference herein. In the present application, the pseudotarget may be, for example, a known amount of a Trichomonas tenax 18SrRNA transcript that can be amplified with a set of amplificationoligonucleotides of the present invention under amplificationconditions, but which does not contain or result in a sequence that isdetectable with a detection probe of the present invention.

D. Sample Processing

Sample processing prior to amplification or detection of a targetsequence may be necessary or useful for discriminating a target sequencefrom non-target nucleic acid present in a sample. Sample processingprocedures may include, for example, direct or indirect immobilizationof nucleic acids and/or oligonucleotides from the liquid phase in aheterogeneous assay. With some procedures, such immobilization mayrequire multiple hybridization events. Ranki et al., “Detection ofMicrobial Nucleic Acids by a One-Step Sandwich Hybridization Test,” U.S.Pat. Nos. 4,486,539 and 4,563,419, for example, disclose a one-stepnucleic acid sandwich” hybridization method involving the use of asolid-phase bound nucleic acid having a target complementary sequenceand a labeled nucleic acid probe which is complementary to a distinctregion of the target nucleic acid. Stabinsky, “Methods and Kits forPerforming Nucleic Acid Hybridization Assays,” U.S. Pat. No. 4,751,177,discloses methods including a “mediator” polynucleotide that reportedlyovercomes sensitivity problems associated with Ranki's method resultingfrom leakage of immobilized probe from the solid support. Instead ofdirectly immobilizing the target nucleic acid, the mediatorpolynucleotides of Stabinsky are used to bind and indirectly immobilizetarget polynucleotide:probe polynucleotide complexes which have formedfree in solution.

Any known solid support may be used for sample processing, such asmatrices and particles free in solution. The solid support may be, forexample, nitrocellulose, nylon, glass, polyacrylate, mixed polymers,polystyrene, silane polypropylene and, preferably, particles having amagnetic charge to facilitate recovering sample and/or removing unboundnucleic acids or other sample components. Particularly preferredsupports are magnetic spheres that are monodisperse (i.e., uniform insize ±5%), thereby providing consistent results, which is particularlyadvantageous for use in an automated procedure. One such automatedprocedure is disclosed by Ammann et al., “Automated Process forIsolating and Amplifying a Target Nucleic Acid Sequence,” U.S. Pat. No.6,335,166.

An oligonucleotide for immobilizing a target nucleic acid on a solidsupport may be joined directly or indirectly to the solid support by anylinkage or interaction which is stable under assay conditions (e.g.,conditions for amplification and/or detection). Referred to herein as an“immobilized probe,” this oligonucleotide may bind directly to thetarget nucleic acid or it may include a base sequence region, such as ahomopolymeric tract (e.g., a poly dT) or a simple short repeatingsequence (e.g., an AT repeat), which hybridizes to a complementary basesequence region present on a capture probe. Directjoining occurs whenthe immobilized probe is joined to the solid support in the absence ofan intermediate group. For example, directjoining may be via a covalentlinkage, chelation or ionic interaction. Indirect joining occurs whenthe immobilized probe is joined to the solid support by one or morelinkers. A “linker” is a means for binding at least two differentmolecules into a stable complex and contains one or more components of abinding partner set.

Members of a binding partner set are able to recognize and bind to eachother. Binding partner sets may be, for example, receptor and ligand,enzyme and substrate, enzyme and cofactor, enzyme and coenzyme, antibodyand antigen, sugar and lectin, biotin and streptavidin, ligand andchelating agent, nickel and histidine, substantially complementaryoligonucleotides, and complementary homopolymeric nucleic acids orhomopolymeric portions of polymeric nucleic acids. Components of abinding partner set are the regions of the members that participate inbinding.

A preferred sample processing system having practical advantages interms of its ease of use and rapidity comprises an immobilized probecontaining a base sequence which is complementary to a base sequence ofa capture probe, referred to herein as an “immobilized probe bindingregion.” The capture probe additionally contains a base sequence,referred to herein as a “target binding region,” which may specificallyhybridize to a target sequence contained in a target nucleic acid underassay conditions. (While specificity of the target binding region of thecapture probe for a region of the target nucleic acid is desirable tominimize the number of non-target nucleic acids remaining from thesample after a separation step, it is not a requirement of the captureprobes of the present invention if the capture probes are being usedsolely to isolate target nucleic acid.) If the capture probe is notbeing employed to isolate a target nucleic acid for subsequentamplification of a target sequence, the capture probe may furtherinclude a detectable label attached within or near the target bindingregion, such as a substituted or unsubstituted acridinium ester. Thelabeled capture probe may be used in a homogeneous or semi-homogenousassay to specifically detect hybrid nucleic acids without detectingsingle-stranded nucleic acids, such as the capture probe. A preferredhomogenous assay which could be used with this system is thehybridization protection assay (HPA), which is discussed above in thesection entitled “Hybridization Conditions and Probe Design.” Followingthe HPA format, label associated with capture probes which have nothybridized to target nucleic acids would be hydrolyzed with the additionof a mild base, while label associated with capture probe:target hybridswould be protected from hydrolysis.

An advantage of this latter assay system is that only a singletarget-specific hybridization event (capture probe:target) is necessaryfor target detection, rather than multiple such events (e.g., captureprobe:target and probe:target or probe:amplicon) which are required inother sample processing procedures described herein. Also, feweroligonucleotides in an assay tend to make the assay faster and simplerto optimize, since the overall rate at which a target nucleic acid iscaptured and detected is limited by the slowest hybridizingoligonucleotide. While the target binding region of a capture probe maybe less specific in alternative assay systems, it must still be rareenough to avoid significant saturation of the capture probe withnon-target nucleic acids. Thus, the requirement that two separate andspecific target sequences be identified in these alternative systemscould place constraints on the identification of an appropriate target.By contrast, only one such target sequence is needed when the captureprobe simultaneously functions as the detection probe.

Whichever approach is adopted, the assay needs to include means fordetecting the presence of the target nucleic acid in the test sample. Avariety of means for detecting target nucleic acids are well known tothose skilled in the art of nucleic acid detection, including meanswhich do not require the presence of a detectable label. Nevertheless,probes including a detectable label are preferred. A labeled probe fordetecting the presence of a target nucleic acid would have to include abase sequence which is substantially complementary and specificallyhybridizes to a target sequence contained in the target nucleic acid.Once the probe stably binds to the target nucleic acid, and theresulting target:probe hybrid has been directly or indirectlyimmobilized, unbound probe can be washed away or inactivated and theremaining bound probe can be detected and/or measured.

Preferred sample processing systems combine the elements of detectionand nucleic acid amplification. These systems first directly orindirectly immobilize a target nucleic acid using a capture probe, thecaptured target nucleic acid is purified by removing inter alia cellulardebris, non-target nucleic acid and amplification inhibitors from thesample-containing vessel, which is followed by amplification of a targetsequence contained in the target nucleic acid. Amplified product is thendetected, preferably in solution with a labeled probe. (The targetnucleic acid may remain in the immobilized state during amplification orit may be eluted from the solid support prior to amplification usingappropriate conditions, such as by first incubating at a temperatureabove the T_(m) of the capture probe:target complex and/or the T_(m) ofthe capture probe:immobilized probe complex.) A preferred embodiment ofthis system is disclosed by Weisburg et al., “Two-Step Hybridization andCapture of a Polynucleotide,” U.S. Pat. No. 6,110,678. In this system,the capture probe hybridizes to the target nucleic acid and animmobilized probe hybridizes to the capture probe:target complex underdifferent hybridization conditions. Under a first set of hybridizationconditions, hybridization of the capture probe to the target nucleicacid is favored over hybridization of the capture probe to theimmobilized probe. Thus, under this first set of conditions, the captureprobe is in solution rather than bound to a solid support, therebymaximizing the concentration of the free capture probe and utilizingfavorable liquid phase kinetics for hybridization to the target nucleicacid. After the capture probe has had sufficient time to hybridize tothe target nucleic acid, a second set of hybridization conditions isimposed permitting in the capture probe:target complex to hybridize tothe immobilized probe, thereby isolating the target nucleic acid in thesample solution. The immobilized target nucleic acid may then bepurified, and a target sequence present in the target nucleic acid maybe amplified and detected. A purification procedure which includes oneor more wash steps is generally desirable when working with crudesamples (e.g., clinical samples) to prevent enzyme inhibition and/ornucleic acid degradation due to substances present in the sample.

A preferred amplification method is the transcription-mediatedamplification method disclosed by Kacian et al., “Nucleic Acid SequenceAmplification Methods,” U.S. Pat. No. 5,480,789. In accord with thismethod, a promoter-primer having a 3′ region complementary to a portionof the target and a 5′ promoter region and a primer having the samenucleotide sequence as a portion of the target are contacted with atarget RNA molecule. The primer and promoter-primer define theboundaries of the target region to be amplified, including both thesense present on the target molecule and its complement, and thus thelength and sequence of the amplicon.

In this preferred embodiment, the amplification oligonucleotides andimmobilized target RNA are contacted in the presence of effectiveamounts of Moloney murine leukemia virus-derived reverse transcriptaseand T7 RNA polymerase, both ribonucleotide and deoxyribonucleotidetriphosphates, and necessary salts and cofactors at 42° C. Under theseconditions, nucleic acid amplification occurs, resulting predominantlyin the production of RNA amplicons of a sense opposite to that of thetarget nucleic acid. These amplicons can then be detected in solutionby, for example, using an acridinium ester-labeled hybridization assayprobe of the same sense as the target nucleic acid, employing HPA, asdisclosed by Arnold et al. in U.S. Pat. No. 5,283,174.

The 3′ terminus of the immobilized probe and the capture probe arepreferably “capped” or blocked to prevent or inhibit their use astemplates for nucleic acid polymerase activity. Capping may involveadding 3′ deoxyribonucleotides (such as cordycepin),3′,2′-dideoxynucleotide residues, non-nucleotide linkers, such as thosedisclosed by Arnold et al. in U.S. Pat. No. 6,031,091, alkane-diolmodifications, or non-complementary nucleotide residues at the 3′terminus.

Those skilled in the art will recognize that the above-describedmethodology is amenable, either as described or with obviousmodifications, to various other amplification schemes, including, forexample, the polymerase chain reaction (PCR), Qβreplicase-mediatedamplification, self-sustained sequence replication (3SR), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), loop-mediated isothermal amplification (LAMP),and the ligase chain reaction (LCR).

E. Capture Probes for Isolating Trichomonas vaginalis Ribosomal NucleicAcid

Capture probes of the present invention are designed to bind to andisolate nucleic acid derived from the 18S ribosomal nucleic acid of T.vaginalis in the presence of non-target nucleic acid. As such, thecapture probes preferably include both a target binding region and animmobilized probe binding region. The target binding region of thecapture probes includes a base sequence which hybridizes to a targetsequence derived from 18S ribosomal nucleic acid from T. vaginalis underassay conditions. While not essential, the target binding regionpreferably exhibits specificity for the target sequence in the presenceof non-target nucleic acid under assay conditions. The immobilized probebinding region has a base sequence which hybridizes to an immobilizedprobe comprising a polynucleotide, or a chimeric containingpolynucleotide sequences, which is joined to a solid support present inthe test sample, either directly or indirectly. The target bindingregion and the immobilized probe binding region may be joined to eachother directly or by means of, for example, a nucleotide base sequence,an abasic sequence or a non-nucleotide linker.

In a preferred embodiment, capture probes according to the presentinvention include a target binding region having a base sequence regionwhich comprises, overlaps with, consists essentially of, consists of,substantially corresponds to, or is contained within the base sequenceof SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32. Theimmobilized probe binding region of these preferred capture probescomprises a base sequence which hybridizes to an immobilized probejoined directly or indirectly to a solid support provided to the testsample under assay conditions. Preferably, the immobilized probe bindingregion comprises a homopolymeric region (e.g., poly dA) located at the3′ end of the capture probe which is complementary to a homopolymericregion (e.g., poly dT) located at the 5′ end of the immobilized probe.The immobilized probe binding region preferably consists of the basesequence of SEQ ID NO:98 tttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. Other basesequences may be incorporated into the immobilized probe binding region,including, for example, short repeating sequences.

To prevent undesirable cross-hybridization reactions, the capture probesof the present invention preferably exclude nucleotide base sequences,other than the nucleotide base sequence of the target binding region,which can stably bind to nucleic acid derived from any organism whichmay be present in the test sample under assay conditions. Consistentwith this approach, and in order to maximize the immobilization ofcapture probe:target complexes which are formed, the nucleotide basesequence of the immobilized probe binding region is preferably designedso that it can stably bind to a nucleotide base sequence present in theimmobilized probe under assay conditions and not to nucleic acid derivedfrom any organism which may be present in the test sample.

The target binding region and the immobilized probe binding region ofthe capture probe may be selected so that the capture probe:targetcomplex has a higher T_(m) than the T_(m) of the captureprobe:immobilized probe complex. In this way, a first set of conditionsmay be imposed which favors hybridization of the capture probe to thetarget sequence over the immobilized probe, thereby providing foroptimal liquid phase hybridization kinetics for hybridization of thecapture probe to the target sequence. Once sufficient time has passedfor the capture probe to bind to the target sequence, a second set ofless stringent conditions may be imposed which allows for hybridizationof the capture probe to the immobilized probe.

Capture probes of the present invention may also include a label or apair of interacting labels for direct detection of the target sequencein a test sample. Non-limiting examples of labels, combinations oflabels and means for labeling probes are set forth supra in the sectionentitled “Preparation of Oligonucleotides” and infra in the sectionentitled “Detection Probes to Trichomonas vaginalis Ribosomal NucleicAcid.” A particularly useful method for detecting the presence of acapture probe hybridized to a target nucleic acid is the HybridizationProtection Assay (HPA), which is described above in the section entitled“Hybridization Conditions and Probe Design.” HPA is a homogenous assaywhich distinguishes between probe hybridized to target nucleic acid andprobe which remains unhybridized. Signal detected from an HPA reactionvessel provides an indication of the presence or amount of targetorganisms in the test sample.

Despite their application in a direct detection assay, the most commonuse of capture probes is in the isolation and purification of targetnucleic acid prior to amplifying a target sequence contained in thetarget nucleic acid. By isolating and purifying the target nucleic acidprior to amplification, the number of unintended amplification reactions(i.e., amplification of non-target nucleic acid) can be severelylimited. And, to prevent or inhibit the capture probe itself fromfunctioning as a template for nucleic acid polymerase activity in thepresence of amplification reagents and under amplification conditions,the 3′ end of the capture probe may be capped or blocked. Examples ofcapping agents include 3′ deoxyribonucleotides, 3′,2′-dideoxynucleotideresidues, non-nucleotide linkers, alkane-diol modifications, andnon-complementary nucleotide residues at the 3′ terminus.

F. Detection Probes to Trichomonas vaginalis Ribosomal Nucleic Acid

This embodiment of the invention relates to novel detection probes.Hybridization is the association of two single strands of complementarynucleic acid to form a hydrogen-bonded double strand. A nucleic acidsequence able to hybridize to a nucleic acid sequence sought to bedetected (“target sequence”) can serve as a probe for the targetsequence. Hybridization may occur between complementary nucleic acidstrands, including DNA/DNA, DNA/RNA, and RNA/RNA, as well as betweensingle-stranded nucleic acids wherein one or both strands of theresulting hybrid contain at least one modified nucleotide, nucleoside,nucleobase, and/or base-to-base linkage. In any case, two single strandsof sufficient complementarity may hybridize to form a double-strandedstructure in which the two strands are held together by hydrogen bondsbetween pairs of complementary bases. As described above, in general Ais hydrogen-bonded to T or U, while G is hydrogen-bonded to C. At anypoint along the hybridized strands, therefore, the classical base pairsAT or AU, TA or UA, GC, or CG may be found. Thus, when a first singlestrand of nucleic acid contains sufficient contiguous complementarybases to a second, and those two strands are brought together underconditions that promote their hybridization, double-stranded nucleicacid will result. Accordingly, under appropriate conditions,double-stranded nucleic acid hybrids may be formed.

The rate and extent of hybridization is influenced by a number offactors. For instance, it is implicit that if one of the two strands iswholly or partially involved in a hybrid, it will be less able toparticipate in the formation of a new hybrid. By designing a probe sothat a substantial portion of the sequence of interest issingle-stranded, the rate and extent of hybridization may be greatlyincreased. Also, if the target is an integrated genomic sequence it willnaturally occur in a double-stranded form, as is the case with a productof PCR. These double-stranded targets are naturally inhibitory tohybridization with a single-stranded probe and require denaturation (inat least the region to be targeted by the probe) prior to thehybridization step. In addition, there can be intra-molecular andinter-molecular hybrids formed within a probe if there is sufficientself-complementarity. Regions of the nucleic acid known or expected toform strong internal structures inhibitory to hybridization are lesspreferred. Examples of such structures include hairpin loops. Likewise,probes with extensive self-complementarity generally should be avoided.All these undesirable structures can be avoided through careful probedesign, and commercial computer programs are available to search forthese types of interactions, such as the Oligo Tech analysis software.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. Molecular torch probesare a type of self-complementary probes that are disclosed by Becker etal., “Molecular Torches,” U.S. Pat. No. 6,361,945. The molecular torchprobes disclosed Becker et al. have distinct regions ofself-complementarity, referred to as “the target binding domain” and“the target closing domain,” which are connected by ajoining region andwhich hybridize to one another under predetermined hybridization assayconditions. When exposed to denaturing conditions, the complementaryregions (which may be fully or partially complementary) of the moleculartorch probe melt, leaving the target binding domain available forhybridization to a target sequence when the predetermined hybridizationassay conditions are restored. And when exposed to strand displacementconditions, a portion of the target sequence binds to the target bindingdomain and displaces the target closing domain from the target bindingdomain. Molecular torch probes are designed so that the target bindingdomain favors hybridization to the target sequence over the targetclosing domain. The target binding domain and the target closing domainof a molecular torch probe include interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch probe is self-hybridized as opposed to when themolecular torch probe is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label or labelsassociated therewith.

Another example of detection probes having self-complementarity are themolecular beacon probes disclosed by Tyagi et al. in U.S. Pat. No.5,925,517. Molecular beacon probes include nucleic acid molecules havinga target complement sequence, an affinity pair (or nucleic acid arms)holding the probe in a closed conformation in the absence of a targetnucleic acid sequence, and a label pair that interacts when the probe isin a closed conformation. Hybridization of the target nucleic acid andthe target complement sequence separates the members of the affinitypair, thereby shifting the probe to an open confirmation. The shift tothe open confirmation is detectable due to reduced interaction of thelabel pair, which may be, for example, a fluorophore and quencher, suchas DABCYL and EDANS.

The rate at which a probe hybridizes to its target is one measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C_(o)t_(1/2),which is measured as moles of nucleotide per liter times seconds. Thus,it is the concentration of probe times the time at which 50% of maximalhybridization occurs at that concentration. This value is determined byhybridizing various amounts of probe to a constant amount of target fora fixed time. The C_(o)t_(1/2) is found graphically by standardprocedures. The probe:target hybrid melting temperature may bedetermined by isotopic methods well-known to those skilled in the art.The melting temperature (T_(m)) for a given hybrid will vary dependingon the hybridization solution being used.

Preferred detection probes are sufficiently complementary to the targetnucleic acid sequence, or its complement, to hybridize therewith understringent hybridization conditions corresponding to a temperature ofabout 60° C. when the salt concentration is in the range of about0.6-0.9 M. Preferred salts include lithium chloride, but other saltssuch as sodium chloride and sodium citrate also can be used in thehybridization solution. Examples of high stringency hybridizationconditions are alternatively provided by 0.48 M sodium phosphate buffer,0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA at atemperature of about 60° C., or by 0.6 M LiCl, 1% lithium lauryl sulfate(LLS), 60 mM lithium succinate and 10 mM each of EDTA and EGTA at atemperature of about 60° C.

Thus, in a first aspect, the present invention features detection probesable to distinguish T. vaginalis-derived nucleic acid from non-T.vaginalis nucleic acid (e.g., Trichomonas tenax) by virtue of theability of the detection probe to preferentially hybridize to T.vaginalis-derived nucleic acid) under stringent hybridizationconditions. Specifically, the detection probes contain anoligonucleotide having a base sequence that is substantiallycomplementary to a target sequence present in T. vaginalis-derivednucleic acid.

In the case of a hybridization assay, the length of the target nucleicacid sequence and, accordingly, the length of the probe sequence can beimportant. In some cases, there may be several sequences from aparticular region, varying in location and length, which will yieldprobes with the desired hybridization characteristics. In other cases,one sequence may have better hybridization characteristics than anotherthat differs merely by a single base. While it is possible for nucleicacids that are not perfectly complementary to hybridize, the longeststretch of perfectly homologous base sequence will normally primarilydetermine hybrid stability. While probes of different lengths and basecomposition may be used, the probes preferred in the present inventionare up to 100 bases in length, more preferably from 12 to 50 bases inlength, and even more preferably from 18 to 35 bases in length.

The detection probes include a base sequence that is substantiallycomplementary to a target sequence present in 18S ribosomal RNA (rRNA),or the encoding DNA (rDNA), of T. vaginalis. Thus, the detection probesare able to stably hybridize to a target sequence derived from T.vaginalis under stringent hybridization conditions. The detection probesmay also have additional bases outside of the targeted nucleic acidregion which may or may not be complementary to T. vaginalis-derivednucleic acid but which are not complementary to nucleic acid derivedfrom a non-target organism which may be present in the test sample.

Probes (and amplification oligonucleotides) of the present invention mayalso be designed to include a capture tail comprised of a base sequence(distinct from the base sequence intended to hybridize to the targetsequence) that can hybridize under predetermined hybridizationconditions to a substantially complementary base sequence present in animmobilized oligonucleotide that is joined to a solid support. Theimmobilized oligonucleotide is preferably joined to a magneticallycharged particle that can be isolated in a reaction vessel during apurification step after a sufficient period of time has passed for probeto hybridize to target nucleic acid. (An example of an instrument whichcan be used to perform such a purification step is the DTS® 1600 TargetCapture System (Gen-Probe; Cat. No. 5202).) The probe is preferablydesigned so that the melting temperature of the probe:target hybrid isgreater than the melting temperature of the probe:immobilizedoligonucleotide hybrid. In this way, different sets of hybridizationassay conditions can be employed to facilitate hybridization of theprobe to the target nucleic acid prior to hybridization of the probe tothe immobilized oligonucleotide, thereby maximizing the concentration offree probe and providing favorable liquid phase hybridization kinetics.This “two-step” target capture method is disclosed by Weisburg et al.,“Two Step Hybridization and Capture of a Polynucleotide,” U.S. Pat. No.6,110,678, the contents of which are hereby incorporated by referenceherein. Other target capture schemes which could be readily adapted tothe present invention are well known in the art and include, forexample, those disclosed by Ranki et al., “Detection of MicrobialNucleic Acids by a One-Step Sandwich Hybridization Test,” U.S. Pat. No.4,486,539, and Stabinsky, “Methods and Kits for Performing Nucleic AcidHybridization Assays,” U.S. Pat. No. 4,751,177.

For T. vaginalis detection probes, the terms “target nucleic acidsequence,” “target nucleotide sequence,” “target sequence,” and “targetregion” all refer to a nucleic acid sequence present in T. vaginalisrRNA or rDNA, or a sequence complementary thereto, which is notidentically present in the nucleic acid of a closely related species.Nucleic acids having nucleotide sequences complementary to a targetsequence may be generated by target amplification techniques disclosedelsewhere herein.

Organisms closely related to T. vaginalis include Trichomonas gallinae,Trichomonas tenax, Monotrichomonas species ATCC 50693, Ditrichomonashonigbergi, Tritrichomonas foetus, Tetratrichomonas gallinarum andPentatrichomonas hominis, with Trichomonas tenax being the most closelyrelated. In addition to these organisms, organisms that might beexpected to be present in a T. vaginalis-containing test sample include,for example, Escherichia coli, Chlamydia trachomatis and Neiserriagonorrhoeae. These lists of organisms are by no means intended to befully representative of the organisms that the T. vaginalis detectionprobes of the present invention can be used to distinguish over. Ingeneral, the T. vaginalis detection probes of the present invention canbe used to distinguish T. vaginalis-derived nucleic acid from any non-T.vaginalis nucleic acid that does not stably hybridize with the probe(s)under stringent hybridization conditions.

In one embodiment, T. vaginalis detection probes of the presentinvention are preferably up to 100 bases in length and comprise a targetbinding region which forms a hybrid stable for detection with a sequencecontained within a target sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. More preferably,the base sequence of the target binding region comprises, overlaps with,consists essentially of, consists of, substantially corresponds to, oris contained within the base sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQID NO:3 or SEQ ID NO:4. In a particularly preferred mode, thesedetection probes include an acridinium ester label joined to the probesby means of a non-nucleotide linker positioned between nucleotides 17and 18 (reading 5′ to 3′) of SEQ ID NO: 1 or SEQ ID NO:2 and betweennucleotides 15 and 16 (reading 5′ to 3′) of SEQ ID NO:3 or SEQ ID NO:4.The acridinium ester label may be joined to the probe in accordance withthe teachings of Arnold et al. in U.S. Pat. Nos. 5,185,439 and6,031,091.

In another embodiment of the present invention, T. vaginalis detectionprobes are preferably up to 100 bases in length and comprise a targetbinding region which forms a hybrid stable for detection with a sequencecontained within a target sequence selected from the group consisting ofSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO: 12 SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 or SEQ ID NO: 16. More preferably, the base sequence of the targetbinding region comprises, overlaps with, consists essentially of,consists of, substantially corresponds to, or is contained within thebase sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:16. One group of preferred T. vaginalisdetection probes has a target binding region comprising, overlappingwith, consisting essentially of, consisting of, substantiallycorresponding to, or contained within the base sequence of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and which may include anacridinium ester label joined to the probe by means of a non-nucleotidelinker positioned between, for example, nucleotides 12 and 13 (reading5′ to 3′) of SEQ ID NO:5 or SEQ ID NO:6 and between, for example,nucleotides 18 and 19 (reading 5′ to 3′) of SEQ ID NO:7 or SEQ ID NO:8.Another group of preferred T. vaginalis detection probes has a targetbinding region comprising, overlapping with, consisting essentially of,consisting of, substantially corresponding to, or contained within thebase sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ IDNO:12, and which may include an acridinium ester label joined to theprobe by means of a non-nucleotide linker positioned between, forexample, nucleotides 17 and 18 (reading 5′ to 3′) of SEQ ID NO:9 or SEQID NO:10 and between, for example, nucleotides 9 and 10 (reading 5′ to3′) of SEQ ID NO:11 or SEQ ID NO:12. A further group of preferred T.vaginalis probes has a target binding region comprising, overlappingwith, consisting essentially of, consisting of, substantiallycorresponding to, or contained within the base sequence of SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, and which may include anacridinium ester label joined to the probe by means of a non-nucleotidelinker positioned between, for example, nucleotides 8 and 9 (reading 5′to 3′) of SEQ ID NO:13 or SEQ ID NO:14 and between, for example,nucleotides 19 and 20 (reading 5′ to 3′) of SEQ ID NO:15 or SEQ IDNO:16. The acridinium ester label may be joined to the probe inaccordance with the teachings of Arnold et al. in U.S. Pat. Nos.5,185,439 and 6,031,091.

Thus, in one aspect of the present invention a detection probe isprovided which is useful for determining whether T. vaginalis is presentin a test sample. The probe is up to 100 bases in length and comprises atarget binding region having a base sequence which comprises, overlapswith, consists essentially of, consists of, substantially correspondsto, or is contained within a base sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 and SEQ ID NO:16. The probe preferentially hybridizes understringent hybridization conditions to a target nucleic acid derived fromT. vaginalis over nucleic acid derived from non-T. vaginalis organismspresent in the test sample. In particular, the probe does not form ahybrid stable for detection with Trichomonas tenax under the stringenthybridization conditions used.

Once synthesized, the probes may be labeled with a detectable label orreporter group by any well-known method. (See, e.g., SAMBROOK ET AL.,supra, ch. 10.) The probe may be labeled with a detectable moiety suchas a radioisotope, antigen or chemiluminescent moiety to facilitatedetection of the target sequence. Useful labels include radioisotopes aswell as non-radioactive reporting groups. Isotopic labels include ³H,³⁵S, ³²P, ¹²⁵I, ⁵⁷Co and ¹⁴C. Isotopic labels can be introduced into anoligonucleotide by techniques known in the art such as nick translation,end labeling, second strand synthesis, reverse transcription and bychemical methods. When using radiolabeled probes, hybridization can bedetected by techniques such as autoradiography, scintillation countingor gamma counting. The chosen detection method depends on the particularradioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally between nucleotides or at an end of theoligonucleotide. Modified nucleotides may be incorporated enzymaticallyor chemically. Chemical modifications of the oligonucleotide may beperformed during or after synthesis of the oligonucleotide usingtechniques known in the art. For example, through use of non-nucleotidelinker groups disclosed by Arnold et al. in U.S. Pat. No. 6,031,091.Non-isotopic labels include fluorescent molecules, chemiluminescentmolecules, fluorescent chemiluminescent molecules, phosphorescentmolecules, electrochemiluminescent molecules, chromophores, enzymes,enzyme cofactors, enzyme substrates, dyes and haptens or other ligands.Another useful labeling technique is a base sequence that is unable tostably hybridize to the target nucleic acid under stringent conditions.Probes of the present invention are preferably labeled with anacridinium ester. (Acridinium ester labeling is disclosed by Arnold etal. in U.S. Pat. No. 5,185,439.)

The selected detection probe can then be brought into contact with atest sample suspected of containing T. vaginalis. Generally, the testsample is from a source that also contains unknown organisms. Typically,the source of the test sample will be a patient specimen, such as agenitourinary specimen. After bringing the probe into contact withnucleic acids derived from the test sample, the probe and sample-derivednucleic acids can be incubated under conditions permitting preferentialhybridization of the probe to a target nucleic acid derived from T.vaginalis that may be present in the test sample in the presence ofnucleic acid derived from other organisms present in the test sample.

Detection probes may also be combined with one or more unlabeled helperprobes to facilitate binding to target nucleic acid derived from T.vaginalis. After a detection probe has hybridized to target nucleic acidpresent in the test sample, the resulting hybrid may be separated anddetected by various techniques well known in the art, such ashydroxyapatite adsorption and radioactive monitoring. Other techniquesinclude those which involve selectively degrading label associated withunhybridized probe and then measuring the amount of remaining labelassociated with hybridized probe, as disclosed in U.S. Pat. No.5,283,174. The inventors particularly prefer this latter technique.

G. Helper Probes Used in the Detection of Trichomonas vaginalis

Another embodiment of this invention relates to novel helper probes. Asmentioned above, helper probes can be used to facilitate hybridizationof detection probes to their intended target nucleic acids, so that thedetection probes more readily form probe:target nucleic acid duplexesthan they would in the absence of helper probes. (Helper probes aredisclosed by Hogan et al., “Means and Method for Enhancing Nucleic AcidHybridization,” U.S. Pat. No. 5,030,557.) Each helper probe contains anoligonucleotide that is sufficiently complementary to a target nucleicacid sequence to form a helper probe:target nucleic acid duplex understringent hybridization conditions. The stringent hybridizationconditions employed with a given helper probe are determined by theconditions used for preferentially hybridizing the associated detectionprobe to the target nucleic acid.

Regions of single-stranded RNA and DNA can be involved in secondary andtertiary structures even under stringent hybridization conditions. Suchstructures can sterically inhibit or block hybridization of a detectionprobe to a target nucleic acid. Hybridization of the helper probe to thetarget nucleic acid alters the secondary and tertiary structure of thetarget nucleic acid, thereby rendering the target region more accessibleby the detection probe. As a result, helper probes enhance the kineticsand/or the melting temperature of the detection probe:target nucleicacid duplex. Helper probes are generally selected to hybridize tonucleic acid sequences located near the target region of the detectionprobe.

Helper probes which can be used with the T. vaginalis detection probesof the present invention are targeted to nucleic acid sequences withinT. vaginalis-derived nucleic acid. Likewise, helper probes which can beused with the T. vaginalis detection probes of the present invention aretargeted to nucleic acid sequences within T. vaginalis-derived nucleicacid. Each helper probe comprises an oligonucleotide which targets andstably hybridizes to a base region present in nucleic acid derived fromT. vaginalis under stringent hybridization conditions. Helper probes andtheir associated detection probes have different target sequencescontained within the same target nucleic acid. The helper probes of thepresent invention are preferably oligonucleotides up to 100 bases inlength, more preferably from 12 to 50 bases in length, and even morepreferably from 18 to 35 bases in length.

Preferred T. vaginalis helper probes useful in the present inventionhave a base sequence comprising, overlapping with, consistingessentially of, consisting of, substantially corresponding to, orcontained within the base sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ IDNO:28. The preferred T. vaginalis detection probe for use with one ormore of these helper probes has a target binding region comprising,overlapping with, consisting essentially of, consisting of,substantially corresponding to, or contained within the base sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, where thedetection probe preferentially hybridizes under stringent hybridizationconditions to a target nucleic acid derived from T. vaginalis overnucleic acid derived from non-T. vaginalis organisms present in a testsample. In particular, the probe does not form a hybrid stable fordetection with Trichomonas tenax under the stringent hybridizationconditions used.

H. Nucleic Acid Composition

In another related aspect, the present invention features compositionscomprising a nucleic acid hybrid formed between a detection probe and atarget nucleic acid (“probe:target”) under stringent hybridizationconditions. One use of the hybrid formed between a probe and a targetnucleic acid is to provide an indication of the presence or amount of atarget organism or group of organisms in a test sample. For example,acridinium ester (AE) present in nucleic acid hybrids is resistant tohydrolysis in an alkali solution, whereas AE present in single-strandednucleic acid is susceptible to hydrolysis in an alkali solution (seeU.S. Pat. No. 5,238,174). Thus, the presence of target nucleic acids canbe detected, after the hydrolysis of the unbound AE-labeled probe, bymeasuring chemiluminescence of acridinium ester remaining associatedwith the nucleic acid hybrid.

The present invention also contemplates compositions comprising nucleicacid hybrids formed between a capture probe and a target nucleic acid(“capture probe:target”) under stringent hybridization conditions. Oneuse of the hybrid formed between a capture probe and a target nucleicacid is to isolate and purify the target nucleic acid in a test sampleprior to amplification of a target sequence contained in the targetnucleic acid or detection of the target nucleic acid in, for example, aheterogenous assay. By isolating and purifying target nucleic acid priorto amplification or detection, the opportunities for non-specificbinding or amplification are significantly minimized.

The present invention further contemplates compositions comprisingnucleic acid hybrids formed between a helper probe and a target nucleicacid (“helper probe:target”) under stringent hybridization conditions.One use of the hybrid formed between a helper probe and a target nucleicacid is to make available a particular nucleic acid sequence forhybridization. For example, a hybrid formed between a helper probe and atarget nucleic acid may render a nucleic acid sequence available forhybridization with a hybridization assay probe. A full description ofthe use of helper probes is provided by Hogan et al. in U.S. Pat. No.5,030,557.

The present invention also features compositions comprising a nucleicacid formed between an amplification oligonucleotide and a targetnucleic acid (“amplification oligonucleotide:target”) underamplification conditions. One use of the hybrid formed between a primerand a target nucleic acid is to provide an initiation site for a nucleicacid polymerase at the 3′ end of the amplification oligonucleotide. Forexample, a hybrid may form an initiation site for reverse transcriptase,DNA polymerases such as Taq polymerase or T4 DNA polymerase, and RNApolymerases such as T7 polymerase, SP6 polymerase, T3 polymerase, andthe like.

Compositions of the present invention include compositions fordetermining the presence or amount of T. vaginalis in a test samplecomprising a nucleic acid hybrid formed between a target nucleic acidderived from T. vaginalis and one or more oligonucleotides, where thebase sequence of each oligonucleotide comprises, overlaps with, consistsessentially of, consists of, substantially corresponds to, or iscontained within the base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 or SEQ ID NO:88. Theoligonucleotides of these compositions may include at least oneadditional nucleotide base sequence region which does not stably bind tonucleic acid derived from T. vaginalis under stringent hybridizationconditions. In another embodiment, the probe:target compositions mayfurther comprise at least one helper probe hybridized to the T.vaginalis-derived target nucleic acid.

The present invention also contemplates compositions for determining thepresence or amount of T. vaginalis in a test sample comprising a nucleicacid hybrid formed between a target nucleic acid derived from T.vaginalis and a detection probe, where the base sequence of thedetection probe comprises, overlaps with, consists essentially of,consists of, substantially corresponds to, or is contained within thebase sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 or SEQ ID NO:16.

Also contemplated by the present invention are compositions forimmobilizing a target nucleic acid derived from a T. vaginalis presentin a test sample comprising a nucleic acid hybrid formed between thetarget nucleic acid and a capture probe comprising a target bindingregion which comprises, overlaps with, consists essentially of, consistsof, substantially corresponds to, or is contained within the basesequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32. Ina further embodiment, these compositions additionally include a nucleicacid hybrid formed between an immobilized probe binding region of thecapture probe and an immobilized probe.

The present invention also contemplates compositions for amplifying atarget sequence present in a target nucleic acid derived from T.vaginalis, where the compositions comprise a nucleic acid hybrid formedbetween the target nucleic acid and an amplification oligonucleotide,where the base sequence of the amplification oligonucleotide comprises,overlaps with, consists essentially of, consists of, substantiallycorresponds to, or consists of the base sequence of SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87 or SEQ ID NO:88. The amplificationprimer of these compositions optionally includes a 5′ sequence which isrecognized by a RNA polymerase or which enhances initiation orelongation by a RNA polymerase. When included, a T7 promoter, such asthe nucleotide base sequence of SEQ ID NO:89, is preferred.

I. Assay Methods

The present invention contemplates various methods for assaying for thepresence or amount of nucleic acid derived from T. vaginalis in a testsample. One skilled in the art will understand that the exact assayconditions, probes, and/or amplification oligonucleotides used will varydepending on the particular assay format used and the source of thesample.

One aspect of the present invention relates to a method for determiningthe presence or amount of T. vaginalis in a test sample by contactingthe test sample under stringent hybridization conditions with adetection probe capable of preferentially hybridizing under stringenthybridization hybridization conditions to a T. vaginalis-derived targetnucleic acid over nucleic acids from non-T. vaginalis organisms presentin the test sample. In such methods, the target nucleic acid contains abase sequence having or substantially corresponding to the base sequenceof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ IDNO:16. (Depending on the source, the test sample may contain unknownorganisms that the probes of this method can distinguish over.) The basesequence of a preferred probe for use in this method comprises, overlapswith, consists essentially of, consists of, substantially correspondsto, or is contained within the base sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

In one preferred embodiment, the method for determining the presence oramount of T. vaginalis in a test sample may also include the step ofcontacting the test sample with one or more helper probes forfacilitating hybridization of the probe to the target nucleic acid.While the helper probes may be added to the sample before or after theaddition of the detection probe, the helper probes and detection probeare preferably provided to the test sample at the same time. The basesequence of a preferred helper probe for use in this method comprises,overlaps with, consists essentially of, consists of, substantiallycorresponds to, or is contained within the base sequence of SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27 or SEQ ID NO:28, and is used in combination with adetection probe, where the base sequence of the detection probecomprises, overlaps with, consists essentially of, consists of,substantially corresponds to, or is contained within the base sequenceof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and where thedetection probe preferentially hybridizes to T. vaginalis-derivednucleic acid over nucleic acid derived from non-T. vaginalis organismspresent in the test sample under stringent hybridization conditions.

Another aspect of the present invention relates to a method foramplifying T. vaginalis-derived nucleic acid in a test sample bycontacting the test sample under amplification conditions with one ormore amplification oligonucleotides which, when contacted with a nucleicacid polymerase, will bind to or cause elongation through a nucleic acidregion having a base sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:87 or SEQ ID NO:88. The amplification oligonucleotideoptionally includes a nucleic acid sequence recognized by a RNApolymerase or which enhances initiation or elongation by a RNApolymerase. Particular combinations of amplification oligonucleotidesthat can be used in this method are set forth above under the heading“Amplification of Trichomonas vaginalis Ribosomal Nucleic Acid.”

In preferred embodiments, the methods for amplifying T.vaginalis-derived nucleic acid in a test sample further include the stepof contacting the test sample under stringent hybridization conditionswith a detection probe capable of preferentially hybridizing understringent hybridization conditions to an amplified T. vaginalis targetnucleic acid over nucleic acids from non-T. vaginalis organisms presentin the test sample. While the test sample is generally contacted withthe detection probe after a sufficient period for amplification haspassed, the amplification oligonucleotides and detection probe may beadded to the sample in any order, as when the detection probe is aself-hybridizing probe, such as a molecular torch probe discussed supra.This step of contacting the test sample with a detection probe isperformed so that the presence or amount of T. vaginalis in the testsample can be determined. The base sequence of a preferred probe for usein this method comprises, overlaps with, consists essentially of,consists of, substantially corresponds to, or is contained within thebase sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 or SEQ ID NO:16. The detection probes may further include a labelto facilitate detection in the test sample.

In certain preferred embodiments, these methods are carried out with aset of at least two amplification oligonucleotides for amplifying T.vaginalis-derived nucleic acid Preferred sets of amplificationoligonucleotides that can be used in these methods are set forth aboveunder the heading “Amplification of Trichomonas vaginalis RibosomalNucleic Acid.” Still another aspect of the present invention relates toa method for immobilizing a target nucleic acid derived from a T.vaginalis in a test sample which comprises providing to the test samplea capture probe having a target binding region and an immobilized probebinding region under a first set of hybridization conditions permittingthe capture probe to stably bind the target nucleic acid, therebyforming a capture probe:target complex, and a second set ofhybridization conditions permitting the capture probe to stably bind toan immobilized probe in the test sample, thereby forming an immobilizedprobe:capture probe:target complex. The first and second sets ofhybridization conditions may be the same or different and the captureprobe:target complex remains stable under the second set ofhybridization conditions. The target binding region of this captureprobe comprises, overlaps with, consists essentially of, consists of,substantially corresponds to, or consists of the base sequence of SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32. A purifying steppreferably follows the immobilizing step to remove one or morecomponents of the test sample that might interfere with or preventamplification or specific detection of a target sequence contained inthe immobilized target nucleic acid. This method for immobilizing andoptionally purifying a T. vaginalis-derived nucleic may precede any ofthe methods described above for amplifying and/or detecting the presenceof a target nucleic acid derived from T. vaginalis. If a purifying stepis included, the target nucleic acid may be indirectly eluted from theimmobilized probe or directly eluted from the capture probe of theimmobilized probe:capture probe:target complex by altering the sampleconditions prior to amplifying or detecting the target sequence.

J. Diagnostic Systems

The present invention also contemplates diagnostic systems in kit form.A diagnostic system of the present invention may include a kit thatcontains, in an amount sufficient for at least one assay, any of thedetection probes, helper probes, capture probes and/or amplificationoligonucleotides of the present invention in a packaging material.Typically, the kits will also include instructions recorded in atangible form (e.g., contained on paper or an electronic medium, such asa disk, CD-ROM, DVD or video tape) for using the packaged probes and/oramplification oligonucleotides in an amplification and/or detectionassay for determining the presence or amount of T. vaginalis in a testsample.

The various components of the diagnostic systems may be provided in avariety of forms. For example, the required enzymes, the nucleotidetriphosphates, the probes and/or primers may be provided as alyophilized reagent. These lyophilized reagents may be pre-mixed beforelyophilization so that when reconstituted they form a complete mixturewith the proper ratio of each of the components ready for use in theassay. In addition, the diagnostic systems of the present invention maycontain a reconstitution reagent for reconstituting the lyophilizedreagents of the kit. In preferred kits for amplifying target nucleicacid derived from T. vaginalis, the enzymes, nucleotide triphosphatesand required cofactors for the enzymes are provided as a singlelyophilized reagent that, when reconstituted, forms a proper reagent foruse in the present amplification methods. In these kits, a lyophilizedprimer reagent may also be provided. In other preferred kits,lyophilized probe reagents are provided.

Typical packaging materials would include solid matrices such as glass,plastic, paper, foil, micro-particles and the like, capable of holdingwithin fixed limits detection probes, helper probes and/or amplificationoligonucleotides of the present invention. Thus, for example, thepackaging materials can include glass vials used to containsub-milligram (e.g., picogram or nanogram) quantities of a contemplatedprobe or primer, or they can be microtiter plate wells to which probesor primers of the present invention have been operatively affixed, i.e.,linked so as to be capable of participating in an amplification and/ordetection method of the present invention.

The instructions will typically indicate the reagents and/orconcentrations of reagents and at least one assay method parameter thatmight be, for example, the relative amounts of reagents to use peramount of sample. In addition, such specifics as maintenance, timeperiods, temperature and buffer conditions may also be included.

The diagnostic systems of the present invention contemplate kits havingany of the detection probes, helper probes, capture probes and/oramplification oligonucleotides described herein, whether providedindividually or in one of the preferred combinations described above,for use in amplifying and/or determining the presence or amount of T.vaginalis in a test sample.

EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the invention. It is believed that these examplesaccurately reflect the details of experiments actually performed,however, it is possible that some minor discrepancies may exist betweenthe work actually performed and the experimental details set forth belowwhich do not affect the conclusions of these experiments. Skilledartisans will appreciate that these examples are not intended to limitthe invention to the specific embodiments described therein.

1. Organism Lysis

Whole cells in the examples below were chemically lysed in a transportmedium described below in the “Reagents” section. This transport mediumis a detergent-containing buffered solution which, in addition to lysingcells, protects released RNAs by inhibiting the activity of RNAsespresent in a test sample.

2. Oligonucleotide Synthesis

Oligonucleotides featured in the examples below include detectionprobes, helper probes, primers and capture probes. Theseoligonucleotides were synthesized using standard phosphoramiditechemistry, various methods of which are well known in the art. See,e.g., Caruthers et al., Methods in Enzymol., 154:287 (1987). Synthesiswas performed using an Expedite™ 8909 Nucleic Acid Synthesizer (AppliedBiosystems; Foster City, Calif.). The detection probes were alsosynthesized to include a non-nucleotide linker, as disclosed by Arnoldet al. in U.S. Pat. Nos. 5,585,481 and 5,639,604, and labeled with achemiluminescent acridinium ester, as disclosed by Arnold et al. in U.S.Pat. No. 5,185,439.

3. Transcription-Mediated Amplification

Amplification of a target sequence in the following examples was by aTranscription-Mediated Amplification (TMA) procedure disclosed by, forexample, Kacian et al. in U.S. Pat. Nos. 5,399,491 and 5,480,784 and byLEE ET AL., supra, ch. 8. TMA is an isothermal amplification procedurewhich allows for a greater than one billion-fold increase in copy numberof the target sequence using reverse transcriptase and RNA polymerase(see Enzyme Reagents below). A TMA reaction involves converting asingle-stranded target sequence to a double-stranded DNA intermediate byreverse transcriptase in the presence of a pair of amplificationoligonucleotides, one of which has a 5′ RNA polymerase-specific promotersequence. In this embodiment, the DNA intermediate includes adouble-stranded promoter sequence which is recognized by a RNApolymerase and directs transcription of the target sequence intohundreds of copies of RNA. Each of these transcribed RNA molecules, inturn, can be converted to a double-stranded DNA intermediate which isused for producing additional RNA. Thus, the TMA reaction proceedsexponentially. The particulars of the TMA reactions used in thefollowing examples are set forth below.

4. Reagents

Various reagents are identified in the examples below, which include alysis buffer, a target capture reagent, an amplification reagent, aprimer reagent, an enzyme reagent, a hybridization reagent, a selectionreagent, and detection reagents. With the exception of Example 1, theformulations and pH values (where relevant) of these reagents were asfollows.

Lysis Buffer: The “Lysis Buffer” of the following examples contained 15mM sodium phosphate monobasic monohydrate, 15 mM sodium phosphatedibasic anhydrous, 1.0 mM EDTA disodium dihydrate, 1.0 mM EGTA freeacid, and 110 mM lithium lauryl sulfate, pH 6.7.

Target Capture Reagent: The “Target Capture Reagent” of the followingexamples contained 250 mM HEPES free acid dihydrate, 310 mM lithiumhydroxide monohydrate, 1.88 M lithium chloride, 100 mM EDTA free acid, 2M lithium hydroxide to pH 6.4, and 250 μg/ml 1 micron magnetic particlesSera-Mag™ MG-CM Carboxylate Modified (Seradyn, Inc.; Indianapolis, Ind.;Cat. No. 24152105-050450) having oligo(dT)₁₄ covalently bound thereto.

Wash Solution: The “Wash Solution” of the following examples contained10 mM HEPES free acid, 6.5 mM sodium hydroxide, 1 mM EDTA free acid,0.3% (v/v) ethyl alcohol absolute, 0.02% (w/v) methyl paraben, 0.01%(w/v) propyl paraben, 150 mM sodium chloride, 0.1% (w/v) lauryl sulfate,sodium (SDS), and 4 M sodium hydroxide to pH 7.5.

Amplification Reagent: The “Amplification Reagent” was a lyophilizedform of a 3.6 mL solution containing 26.7 mM rATP, 5.0 mM rCTP, 33.3 mMrGTP and 5.0 mM rUTP, 125 mM HEPES free acid, 8% (w/v) trehalosedihydrate, 1.33 mM dATP, 1.33 mM dCTP, 1.33 mM dGTP and 1.33 mM dTTP,and 4 M sodium hydroxide to pH 7.5. The Amplification Reagent wasreconstituted in 9.7 mL of the Amplification Reagent ReconstitutionSolution described below.

Amplification Reagent Reconstitution Solution: The “AmplificationReagent Reconstitution Solution” contained 0.4% (v/v) ethyl alcoholabsolute, 0.10% (w/v) methyl paraben, 0.02% (w/v) propyl paraben, 33 mMKCl, 30.6 mM MgCl₂, 0.003% phenol red.

Primer Reagent: The “Primer Reagent” of the following examples contained1 mM EDTA disodium dihydrate, ACS, 10 mM Trizma® base, and 6Mhydrochloric acid to pH 7.5.

Enzyme Reagent: The “Enzyme Reagent” of the following examples was alyophilized form of a 1.45 mL solution containing 20 mM HEPES free aciddihydrate, 125 mM N-acetyl-L-cysteine, 0.1 mM EDTA disodium dihydrate,0.2% (v/v) TRITON® X-100 detergent, 0.2 M trehalose dihydrate, 0.90RTU/mL Moloney murine leukemia virus (“MMLV”) reverse transcriptase, and0.20 U/mL T7 RNA polymerase, and 4M sodium hydroxide to pH 7.0. (One“unit” or “RTU” of activity is defined as the synthesis and release of5.75 fmol cDNA in 15 minutes at 37° C. for MMLV reverse transcriptase,and for T7 RNA polymerase, one “unit” or “U” of activity is defined asthe production of 5.0 fmol RNA transcript in 20 minutes at 37° C.) TheEnzyme Reagent was reconstituted in 3.6 mL of the Enzyme ReagentReconstitution Solution described below.

Enzyme Reagent Reconstitution Solution: The “Enzyme ReagentReconstitution Solution” of the following examples contained 50 mM HEPESfree acid, 1 mM EDTA free acid, 10% (v/v) TRITON® X-100 detergent, 120mM potassium chloride, 20% (v/v) glycerol anhydrous, and 4 M sodiumhydroxide to pH 7.0.

Hybridization Reagent: The “Hybridization Reagent” contained 100 mMsuccinic acid free acid, 2% (w/v) lithium lauryl sulfate, 100 mM lithiumhydroxide monohydrate, 15 mM aldrithiol-2, 1.2 M lithium chloride, 20 mMEDTA free acid, 3.0% (v/v) ethyl alcohol absolute, and 2M lithiumhydroxide to pH 4.7.

Selection Reagent: The “Selection Reagent” of the following examplescontained 600 mM boric acid, ACS, 182.5 mM sodium hydroxide, ACS, 1%(v/v) TRITON® X-100 detergent, and 4 M sodium hydroxide to pH 8.5.

Detection Reagents: The “Detection Reagents” of the following examplescomprised Detect Reagent I, which contained 1 mM nitric acid and 32 mMhydrogen peroxide, 30% (v/v), and Detect Reagent II, which contained 1.5M sodium hydroxide.

Oil Reagent: The “Oil Reagent” of the following examples was a siliconeoil (United Chemical Technologies, Inc., Bristol, Pa.; Cat. No. PS038).

Example 1 Specificity of T. vaginalis Direct Detection Assay

In this experiment, we compared the specificity of two detection probestargeting different regions of the 18S rRNA of T. vaginalis(ATCC No.50143) in a non-amplified, direct detection assay. The probes of thisexperiment were tested alone or in combination with each other and/or apair of helper probes. For each organism tested, sample tubes wereprepared containing two replicates of each of the approximate cellamounts indicated in Table 2 below. The non-target organisms includedGiardia intestinalis (ATCC No. 30888), Trimastix pyriformis (ATCC No.50562) and Trichomonas tenax(ATCC No. 30207). Two replicates each ofboth a negative control and a T. vaginalis rRNA positive control werealso included to confirm that the reagents and conditions supporteddetectable hybridization of the probes to the target sequences and thatdetectable hybridization would not occur in the absence of the targetnucleic acid. The negative control was also used to determine backgroundsignal. To lyse the cells and release nucleic acid, the contents of eachsample tube were suspended in 300 μL of a lysis buffer (Gen-Probe; Cat.No. 3275 or 3300) and then heated in a 95° C. water bath for about 10minutes. Following incubation, the samples were cooled at roomtemperature for about 5 minutes.

Sample tubes were also set up in the tube rack of a magnetic separationunit (Gen-Probe; Cat. No. 1639) and each was provided with 100 μL of ahybridization reagent (3 mM EDTA disodium dihydrate, 3 mM EGTA freeacid, 17% (w/v) lithium lauryl sulfate, 190 mM succinic acid free acid,lithium hydroxide monohydrate, and 2 M lithium hydroxide to pH 5.1). Thehybridization reagent included one of the probe or probe mix reagents ofTable 1 below, where the amount of probe is indicated by reference to an“RLU” or relative light units value, which is a measure ofchemiluminescence. For these probe and probe mix reagents, Probe 1 hadthe base sequence of SEQ ID NO:7 and a standard acridinium ester labeljoined to the probe by means of a non-nucleotide linker positionedbetween nucleotides 12 and 13 (reading 5′ to 3′), Probe 2 had the basesequence of SEQ ID NO:3 and a standard acridinium ester label joined tothe probe by means of a non-nucleotide linker positioned betweennucleotides 14 and 15 (reading 5′ to 3′), Helper Probe 1 had the basesequence of SEQ ID NO:23, and Helper Probe 2 had the base sequence ofSEQ ID NO:27.

TABLE 1 Probe and Probe Mix Reagents Helper Helper Probe 1 Probe 2 Probe1 Probe 2 (3 × 10⁶ RLU) (3 × 10⁶ RLU) (3 pmol) (3 pmol) Reagent A ✓ ✓ ✓✓ Reagent B ✓ Reagent C ✓ Reagent D ✓ ✓ ✓

After adding the probe and probe mix reagents, the sample tubes werevortexed for about 10 seconds to ensure homogeneity of the hybridizationreagent. The hybridization reagent of each sample tube was combined with100 μL of lysed material from one of the sample tubes above. The sampletubes were then covered with sealing cards (Gen-Probe; Cat. No. 2085)and the rack was hand-shaken several times to mix the contents of thesample tubes prior to incubating the sample tubes in a 60° C. water bathfor about 1 hour.

The tube rack was removed from the water bath and the sealing cards wereremoved from the sample tubes before adding 1 mL of a separationsuspension to each sample tube. The separation suspension was a 20:1mixture of a selection reagent (222 mM 6N hydrochloric acid solution,190 mM sodium tetraborate, 0.01% (v/v) gelatin (fish skin), and 6.43%(v/v) TRITON® X-102 detergent) and a separation reagent (1 mM EDTAdisodium dihydrate, 0.02% (w/v) sodium azide and 1.25 mg/mL BioMag®particles (Polysciences, Inc., Warrington, Pa.; Cat. No. 8-4100T). Thesample tubes were again covered with sealing cards and the tube rack wasvigorously shaken 3 to 5 times to mix the contents before placing it ina 60° C. water bath for about 10 minutes to immobilize nucleic acidpresent in the sample tubes on the BioMag particles. The tube rack wasremoved from the water bath and the tube rack was placed on the base ofthe magnetic separation unit for 5 minutes at room temperature tomagnetically isolate the BioMag particles. With the sealing cardsremoved, the tube rack and base of the magnetic separation unit wereinverted to decant the supernatants of the sample tubes. To removeresidual liquid, the magnetic separation unit was then shaken 2 to 3times and the sample tubes were blotted 3 times for 5 seconds onabsorbent paper. Each sample tube was then filled to the rim with a washsolution (25 mM sodium hydroxide, 20 mM sodium tetraborate, 0.1% (w/v)Zwittergent® 3-14 detergent, and 4M sodium hydroxide to pH 10.4) andallowed to remain on the base of the magnetic separation unit for 20minutes at room temperature. Holding the tube rack and base of themagnetic separation unit together, the supernatants were decanted andthe magnetic separation unit was shaken 2 to 3 times. The tubes werereturned to their upright position, leaving about 50 to 100 μL of thewash solution in each sample tube, and the tube rack was separated fromthe base of the magnetic separation unit. The sample tubes were thenanalyzed in a LEADER® 450 h or a LEADER® HC+ Luminometer equipped withautomatic injection of Detection Reagent 1, followed by automaticinjection of Detection Reagent 2. An RLU value of 1000 was determined tobe the cut-off for a negative result.

The results are summarized in Table 2 below and indicate that the probesand probe mixes tested in this experiment were specific for T.vaginalis.

TABLE 2 Specificity of T. vaginalis Direct Detection Assay Cell Avg. RLUSample Count Reagent A Reagent B Reagent C Reagent D Giardia 2 × 10⁵−189 170 58 315 intestinalis 2 × 10⁴ 191 −10 328 −31 Trimastix 1 × 10⁵31 −186 453 −128 pyriformis 1 × 10⁴ −120 −26 266 −146 Trichomonas 4.9 ×10⁵   −243 328 52 320 tenax 4.9 × 10⁴   −127 126 14 137 Trichomonas 2 ×10⁵ 636,638 298,244 25,439 356,041 vaginalis 2 × 10⁴ 407,087 53,4705,165 299,776 Positive 10.5 ng 85,274 7,317 2,506 55,597 Control RNANegative N/A 282 219 36 651 Control

Example 2 Sensitivity and Specificity of T. vaginalis AmplificationAssay

This experiment was conducted to determine the sensitivity andspecificity of an amplification assay targeting 18S rRNA of T.vaginalis(ATCC No. 50143) in the presence of several closely-related,non-target organisms. The non-target organisms in this experimentincluded Giardia intestinalis (ATCC No. 30888), Trimastix pyriformis(ATCC No. 50562) and Trichomonas tenax(ATCC No. 30207), the latter beingthe most closely related to T. vaginalis. Sample tubes were prepared inreplicates of four for each organism at each of the approximate cellconcentrations indicated in Table 2 below. Two replicates each of a T.vaginalis RNA positive control (5 fg/replicate) and a negative controlwere also prepared. To lyse the cells and release target nucleic acid,400 μL of the Lysis Buffer was added to each sample tube, and the sampletubes were heated for about 10 minutes in a 95° C. water bath. Followingincubation, the samples were cooled at room temperature for about 5minutes.

To separate T. vaginalis target nucleic acid from other componentspresent in the sample tubes, the contents of the sample tubes weretransferred to the reaction tubes of Ten-Tube Units (Gen-Probe; Cat. No.TU0022) and combined with 100 μL of the Target Capture Reagentcontaining 3 pmol of a target capture probe having the sequence of: SEQID NO:99 gcctgctgctacccgtggatattttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. Thiscapture probe includes a 5′ target binding region (SEQ ID NO: 31) and a3′ immobilized probe binding region (SEQ ID NO:98). The TTUs werecovered with a sealing card (Gen-Probe; Cat. No. 2085), hand-shaken,incubated in a 62° C. water bath for about 30 minutes to permithybridization of the target binding region of the capture probe to thetarget nucleic acid, and cooled at room temperature for about 30 minutesto facilitate hybridization of the oligo(dA)₃₀ sequence of theimmobilized probe binding region of the capture probe to oligo(dT)₁₄bound to the magnetic particles. Following cooling of the samples, aDTS® 1600 Target Capture System (Gen-Probe; Cat. No. 5202) was used toisolate and wash the magnetic particles. The DTS 1600 Target CaptureSystem has a test tube bay for positioning TTUs and applying a magneticfield thereto. The TTUs were placed in the test tube bay on the DTS™1600 Target Capture System for about 5 minutes in the presence of themagnetic field to isolate the magnetic particles within the reactiontubes, after which the sample solutions were aspirated from the TTUs.Each tube was then provided with 1 mL of the Wash Solution, covered witha sealing card and vortexed for 10 to 20 seconds to resuspend themagnetic particles. The TTUs were returned to the test tube bay on theDTS® 1600 Target Capture System and allowed to stand at room temperaturefor about 5 minutes before the wash solution was aspirated.

Following the target capture step, 75 μL of the reconstitutedAmplification Reagent spiked with a pair of primers was added to each ofthe reaction tubes. Each primer was present at a concentration of 15pmol in the spiked Amplification Reagent. (It is noted that 4 pmol ofeach primer per reaction mixture is currently preferred.) The primersfor this experiment included a primer having the base sequence of SEQ IDNO:45 and a promoter-primer having the base sequence of SEQ ID NO: 100aatttaatacgactcactatagggagaggcatcacggac ctgttattgc. The promoter primerincluded a 3′ target-binding portion (SEQ ID NO:55) and a 5′ T7 promotersequence (SEQ ID NO: 89). The reaction tubes were then provided with 200μL of the Oil Reagent, covered with a sealing card, and vortexed forabout 10 seconds before being incubated in a 62° C. water bath for about10 minutes for an initial anneal step to promote binding of thepromoter-primers to the target nucleic acid. The reaction tubes weretransferred to a 42° C. water bath for about 5 minutes, the sealingcards were removed from the reaction tubes, and 25 μL of thereconstituted Enzyme Reagent was added to each of the reaction tubes.The reaction tubes were again covered with a sealing card, removed fromthe water bath, and their contents were gently mixed by hand. Aftermixing, the reaction tubes were again incubated in the 42° C. water bathfor about 60 minutes.

For detection of T. vaginalis amplification products, the reaction tubeswere removed from the water bath and 100 μL of the Hybridization Reagentcontaining 100 fmol of a detection probe was added to each reactiontube. The detection probe had the base sequence of SEQ ID NO:3 and astandard acridinium ester label joined to the probe by means of anon-nucleotide linker positioned between nucleotides 17 and 18, reading5′ to 3′. The reaction tubes were covered with a sealing card andvortexed for about 10 seconds before being incubated in a 62° C. waterbath for about 20 minutes to allow hybridization of the detection probeto amplification products present in the reaction tubes. The reactiontubes were then removed from the water bath and allowed to cool at roomtemperature for about 5 minutes before adding 250 μL of the SelectionReagent to each reaction tube. The reaction tubes were covered with asealing card and vortexed for about 10 seconds before being incubated ina 62° C. water bath for about 10 minutes to hydrolyze acridinium esterlabels associated with unhybridized probe. The reaction tubes were thencooled in a 18° to 28° C. water bath for about 15 minutes before beinganalyzed in a LEADER® 450 h or a LEADER® HC+ Luminometer equipped withautomatic injection of Detection Reagent 1, followed by automaticinjection of Detection Reagent 2. The cut-off for a negative result inthis experiment was 50,000 RLU.

The results are summarized in Table 3 below and indicate that the T.vaginalis assay of this experiment amplified and detected T.vaginalis-derived nucleic acid without cross-reacting with nucleic acidderived from Giardia lamblia, Trimastix pyriformis or Trichomonas tenax.As above, the term “RLU” in this table stands for relative light units,and the term “CV” stands for coefficient of variation and represents thestandard deviation of the replicates over the mean of the replicates asa percentage.

TABLE 3 Sensitivity and Specificity of the T. vaginalis AmplificationAssay Sample Cell Count Avg. RLU % CV Giardia 4 × 10⁴ 2925 9 lamblia 4 ×10³ 2882 16 4 × 10² 3100 28 Trimastix 4 × 105 3322 14 pyriformis 4 × 10⁴2720 7 4 × 10³ 2769 19 Trichomonas 4 × 10⁴ 2473 18 tenax 4 × 10³ 4613 714 × 10² 2315 7 Trichomonas 4 × 10⁴ 2,362,258 59 vaginalis 4 × 10³1,427,954 46 4 × 10² 2,754,667 31 Positive Control N/A 4,359,224 2Negative Control N/A 5122 47

Example 3 Primer Sets for Use in a T. vaginalis Amplification AssayDirected to the 400 Region of T. vaginalis 18S rRNA

The purpose of this experiment was to compare the amplificationefficiency of various primer sets for amplifying a portion of the 400region of a transcript derived from the 18S rRNA of T. vaginalis atdifferent initial annealing temperatures. The amplification anddetection procedures of this experiment were identical to those ofExample 2 above, except that one group of primer sets was exposed to an95° C. initial annealing step instead of a 62° C. initial annealing stepafter the Oil Reagent was added to the sample tubes. Because the primersets of this experiment targeted transcript, a target capture step wasnot included.

The detection probe used for detecting the formation of amplificationproducts in this experiment had the base sequence of SEQ ID NO:9 and astandard acridinium ester label joined to the probe by means of anon-nucleotide linker positioned between nucleotides 17 and 18, reading5′ to 3′. Primers 1-3 identified in Tables 3 and 4 below had the basesequences of SEQ ID Nos. 61, 65 and 69, respectively. And Primers 4-6identified in Tables 3 and 4 below were promoter-primers having thefollowing base sequences:

Primer 4: (SEQ ID NO:101)aatttaatacgactcactatagggagacctctgctaggtttcggtacg gt, Primer 5: (SEQ IDNO:102) aatttaatacgactcactatagggagagactggccctctgctaggttt cg, and Primer6: (SEQ ID NO:103) aatttaatacgactcactatagggagagctgctggcaccagactgg.Primers 4-6 had a 5′ promoter sequence (SEQ ID NO:89) and 3′ targetbinding portions having the base sequences of SEQ ID Nos. 79, 83 and 87,respectively.

The results are summarized in Tables 4 and 5 below and indicate that theprimer set of Primers 3 and 5 performed the best at amplifying thetarget region under both sets of conditions. The results also indicatethat in most instances, the amplification efficiency of the primer setswas better with a 95° C. rather than a 62° C. initial annealing step. Asabove, the term “RLU” in these tables stands for relative light units,and the term “CV” stands for coefficient of variation.

TABLE 4 Primer Sets for T. vaginalis rRNA Amplification Employing a 62°C. Initial Annealing Step Primer Set Copy Number Avg. RLU % CV Primers1.5 × 10⁴ 41,788 30 3 and 4 1,875 30,053 23 Primers 1.5 × 10⁴ 53,763 103 and 5 1,875 46,493 13 Primers 1.5 × 10⁴ 3,619 4 1 and 6 1,875 3,176 4Primers 1.5 × 10⁴ 28,059 43 2 and 6 1,875 11,758 90 Primers 1.5 × 10⁴46,365 12 3 and 6 1,875 6,646 13

TABLE 5 Primer Sets for T. vaginalis rRNA Amplification Employing a 95°C. Initial Annealing Step Primer Set Copy Number Avg. RLU % CV Primers1.5 × 10⁴ 59,660 22 3 and 4 1,875 40,248 15 Primers 1.5 × 10⁴ 85,698 533 and 5 1,875 45,099 12 Primers 1.5 × 10⁴ 5,553 65 1 and 6 1,875 11,286157 Primers 1.5 × 10⁴ 20,033 47 2 and 6 1,875 17,104 47 Primers 1.5 ×10⁴ 40,969 8 3 and 6 1,875 13,323 46

Example 4 Primer Sets for Use in a T. vaginalis Amplification AssayDirected to the 1100 Region of T. vaginalis 18S rRNA

The purpose of this experiment was to compare the amplificationefficiency of several primer sets for amplifying a portion of the 1100region of a transcript derived from the 18S rRNA of T. vaginalis. Theamplification and detection procedures of this experiment were identicalto those of Example 2 above. A target capture step was not included.

The detection probe used for detecting the formation of amplificationproducts in this experiment had the base sequence of SEQ ID NO:3 and astandard acridinium ester label joined to the probe by means of anon-nucleotide linker positioned between nucleotides 17 and 18, reading5′ to 3′. Primers 1 and 2 identified in Table 5 below had the basesequences of SEQ ID Nos. 41 and 45, respectively. And Primers 3 and 4identified in Tables 6 below were promoter-primers having the followingbase sequences:

Primer 3: aatttaatacgactcactatagggagacctcttccacctgctaaaatcgcag (SEQ IDNO:104), and

Primer 4: aatttaatacgactcactatagggagaggcatcacggacctgttattgc (SEQ IDNO:105). Primers 3 and 4 had a 5′ promoter sequence (SEQ ID NO: 89) and3′ target binding portions having the base sequences of SEQ ID Nos. 51and 55, respectively.

The results are summarized in Table 6 below and indicate that primersets which included the promoter-primer of SEQ ID NO: 105 (Primer 4)were superior at amplifying the target region. As above, the term “RLU”in this table stands for relative light units, and the term “CV” standsfor coefficient of variation.

TABLE 6 Primer Sets for T. vaginalis rRNA Amplification Primer Set CopyNumber Avg. RLU % CV Primers 1.5 × 10⁴ 306,917 4 1 and 3 1,875 130,84210 Primers 1.5 × 10⁴ 5,262,130 1 1 and 4 1,875 4,584,780 1 Primers 1.5 ×10⁴ 169,767 6 2 and 3 1,875 27,693 16 Primers 1.5 × 10⁴ 5,193,952 3 2and 4 1,875 5,049,133 1

Example 5 Sensitivity of T. vaginalis Amplification Assay

This experiment was designed to evaluate the sensitivity of anamplification assay targeting a portion of the 1100 region of 18S rRNAof T. vaginalis present in a transcript derived from T. vaginalisnucleic acid following the procedures and employing the detection probeand Primers 2 and 4 of Example 4 above. The results of this experimentare summarized in Table 7 below and indicate at least about 19 copysensitivity (one of the replicates had a total RLU of about 300,000, thecut-off for a positive result in this assay). As above, the term “RLU”in this table stands for relative light units, and the term “CV” standsfor coefficient of variation. The CV values are generally larger withlower concentrations of transcript because some of the replicates arebeing amplified, while others were not, thereby resulting in a higherstandard deviation between the replicates.

TABLE 7 Varying Concentrations of Transcript in T. vaginalisAmplification Assay Copy Number Avg. RLU % CV 20,000 5,182,728 2 10,0005,163,317 2 5000 5,400,837 3 2500 5,360,159 1 1250 5,371,082 4 6255,242,293 2 312 5,098,105 2 156 5,017,485 2 78 4,831,400 2 39 4,661,0455 19 2,908,561 63 0 2695 4

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. A method for amplifying a target region of nucleic acid derived fromTrichomonas vaginalis present in a sample, the method comprising thesteps of: (a) contacting the sample with a set of oligonucleotides, theset of oligonucleotides comprising a first amplificationoligonucleotide, the base sequence of the first amplificationoligonucleotide consisting of a target binding region and, optionally, a5′ sequence recognized by an RNA polymerase or which enhances initiationor elongation by an RNA polymerase, wherein the base sequence of thetarget binding region is perfectly complementary to a sequence containedwithin the base sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 orSEQ ID NO:56; and (b) exposing the sample to conditions sufficient toamplify the target region.
 2. The method of claim 1, wherein the basesequence of the target binding region of the first amplificationoligonucleotide is perfectly complementary to a sequence containedwithin the base sequence of SEQ ID NO:54.
 3. The method of claim 1,wherein the base sequence of the target binding region of the firstamplification oligonucleotide is perfectly complementary to the basesequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56. 4.The method of claim 3, wherein the base sequence of the target bindingregion of the first amplification oligonucleotide is perfectlycomplementary to the base sequence of SEQ ID NO:54.
 5. The method ofclaim 1, wherein the first amplification oligonucleotide includes the 5′sequence recognized by an RNA polymerase or which enhances initiation orelongation by an RNA polymerase.
 6. The method of claim 1 furthercomprising a second amplification oligonucleotide, the base sequence ofthe second amplification oligonucleotide consisting of a target bindingregion and, optionally, a 5′ sequence recognized by an RNA polymerase orwhich enhances initiation or elongation by an RNA polymerase, whereinthe base sequence of the target binding region is perfectlycomplementary to a sequence contained within the base sequence of SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.
 7. The method ofclaim 6, wherein the base sequence of the target binding region of thesecond amplification oligonucleotide is perfectly complementary to asequence contained within the base sequence of SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43 or SEQ ID NO:44.
 8. The method of claim 7, whereinthe base sequence of the target binding region of the firstamplification oligonucleotide is perfectly complementary to a sequencecontained within the base sequence of SEQ ID NO:54, and wherein the basesequence of the target binding region of the second amplificationoligonucleotide is perfectly complementary to a sequence containedwithin the base sequence of SEQ ID NO:44.
 9. The method of claim 8,wherein the base sequence of the target binding region of the firstamplification oligonucleotide is perfectly complementary to the basesequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56,and wherein the base sequence of the target binding region of the secondamplification oligonucleotide is perfectly complementary to the basesequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44.10. The method of claim 9, wherein the base sequence of the targetbinding region of the first amplification oligonucleotide is perfectlycomplementary to the base sequence of SEQ ID NO:54, and wherein the basesequence of the target binding region of the second amplificationoligonucleotide is perfectly complementary to the base sequence of SEQID NO:44.
 11. The method of claim 6, wherein the base sequence of thetarget binding region of the second amplification oligonucleotide isperfectly complementary to a sequence contained with the base sequenceof SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48.
 12. Themethod of claim 11, wherein the base sequence of the target bindingregion of the first amplification oligonucleotide is perfectlycomplementary to a sequence contained within the base sequence of SEQ IDNO:54, and wherein the base sequence of the target binding region of thesecond amplification oligonucleotide is perfectly complementary to asequence contained within the base sequence of SEQ ID NO:48.
 13. Themethod of claim 12, wherein the base sequence of the target bindingregion of the first amplification oligonucleotide is perfectlycomplementary to the base sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55 or SEQ ID NO:56, and wherein the base sequence of the targetbinding region of the second amplification oligonucleotide is perfectlycomplementary to the base sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47 or SEQ ID NO:48.
 14. The method of claim 13, wherein the basesequence of the target binding region of the first amplificationoligonucleotide is perfectly complementary to the base sequence of SEQID NO:54, and wherein the base sequence of the target binding region ofthe second amplification oligonucleotide is perfectly complementary tothe base sequence of SEQ ID NO:48.
 15. The method of claim 6, wherein atleast one of the first and second amplification oligonucleotidesincludes the 5′ sequence.